Isolated human kinase proteins, nucleic acid molecules encoding human kinase proteins, and uses thereof

ABSTRACT

The present invention provides amino acid sequences of peptides that are encoded by genes within the human genome, the kinase peptides of the present invention. The present invention specifically provides isolated peptide and nucleic acid molecules, methods of identifying orthologs and paralogs of the kinase peptides, and methods of identifying modulators of the kinase peptides.

RELATED APPLICATIONS

The present application is a continuation-in-part of U.S. Ser. No.09/804,471, filed Mar. 13, 2001 pending.

FIELD OF THE INVENTION

The present invention is in the field of kinase proteins that arerelated to the citron kinase subfamily, recombinant DNA molecules, andprotein production. The present invention specifically provides novelpeptides and proteins that effect protein phosphorylation and nucleicacid molecules encoding such peptide and protein molecules, all of whichare useful in the development of human therapeutics and diagnosticcompositions and methods.

BACKGROUND OF THE INVENTION

Protein Kinases

Kinases regulate many different cell proliferation, differentiation, andsignaling processes by adding phosphate groups to proteins. Uncontrolledsignaling has been implicated in a variety of disease conditionsincluding inflammation, cancer, arteriosclerosis, and psoriasis.Reversible protein phosphorylation is the main strategy for controllingactivities of eukaryotic cells. It is estimated that more than 1000 ofthe 10,000 proteins active in a typical mammalian cell arephosphorylated. The high energy phosphate, which drives activation, isgenerally transferred from adenosine triphosphate molecules (ATP) to aparticular protein by protein kinases and removed from that protein byprotein phosphatases. Phosphorylation occurs in response toextracellular signals (hormones, neurotransmitters, growth anddifferentiation factors, etc), cell cycle checkpoints, and environmentalor nutritional stresses and is roughly analogous to turning on amolecular switch. When the switch goes on, the appropriate proteinkinase activates a metabolic enzyme, regulatory protein, receptor,cytoskeletal protein, ion channel or pump, or transcription factor.

The kinases comprise the largest known protein group, a superfamily ofenzymes with widely varied functions and specificities. They are usuallynamed after their substrate, their regulatory molecules, or some aspectof a mutant phenotype. With regard to substrates, the protein kinasesmay be roughly divided into two groups; those that phosphorylatetyrosine residues (protein tyrosine kinases, PTK) and those thatphosphorylate serine or threonine residues (serine/threonine kinases,STK). A few protein kinases have dual specificity and phosphorylatethreonine and tyrosine residues. Almost all kinases contain a similar250-300 amino acid catalytic domain. The N-terminal domain, whichcontains subdomains I-IV, generally folds into a two-lobed structure,which binds and orients the ATP (or GTP) donor molecule. The larger Cterminal lobe, which contains subdomains VI A-XI, binds the proteinsubstrate and carries out the transfer of the gamma phosphate from ATPto the hydroxyl group of a serine, threonine, or tyrosine residue.Subdomain V spans the two lobes.

The kinases may be categorized into families by the different amino acidsequences (generally between 5 and 100 residues) located on either sideof, or inserted into loops of, the kinase domain. These added amino acidsequences allow the regulation of each kinase as it recognizes andinteracts with its target protein. The primary structure of the kinasedomains is conserved and can be further subdivided into 11 subdomains.Each of the 11 subdomains contains specific residues and motifs orpatterns of amino acids that are characteristic of that subdomain andare highly conserved (Hardie, G. and Hanks, S. (1995) The Protein KinaseFacts Books, Vol 1:7-20 Academic Press, San Diego, Calif.).

The second messenger dependent protein kinases primarily mediate theeffects of second messengers such as cyclic AMP (cAMP), cyclic GMP,inositol triphosphate, phosphatidylinositol, 3,4,5-triphosphate,cyclic-ADPribose, arachidonic acid, diacylglycerol andcalcium-calmodulin. The cyclic-AMP dependent protein kinases (PKA) areimportant members of the STK family. Cyclic-AMP is an intracellularmediator of hormone action in all prokaryotic and animal cells that havebeen studied. Such hormone-induced cellular responses include thyroidhormone secretion, cortisol secretion, progesterone secretion, glycogenbreakdown, bone resorption, and regulation of heart rate and force ofheart muscle contraction. PKA is found in all animal cells and isthought to account for the effects of cyclic-AMP in most of these cells.Altered PKA expression is implicated in a variety of disorders anddiseases including cancer, thyroid disorders, diabetes, atherosclerosis,and cardiovascular disease (Isselbacher, K. J. et al. (1994) Harrison'sPrinciples of internal Medicine, McGraw-Hill, New York, N.Y., pp.416-431, 1887).

Calcium-calmodulin (CaM) dependent protein kinases are also members ofSTK family. Calmodulin is a calcium receptor that mediates many calciumregulated processes by binding to target proteins in response to thebinding of calcium. The principle target protein in these processes isCaM dependent protein kinases. CaM-kinases are involved in regulation ofsmooth muscle contraction (MLC kinase), glycogen breakdown(phosphorylase kinase), and neurotransmission (CaM kinase I and CaMkinase II). CaM kinase I phosphorylates a variety of substratesincluding the neurotransmitter related proteins synapsin I and II, thegene transcription regulator, CREB, and the cystic fibrosis conductanceregulator protein, CFTR (Haribabu, B. et al. (1995) EMBO Journal14:3679-86). CaM II kinase also phosphorylates synapsin at differentsites, and controls the synthesis of catecholamines in the brain throughphosphorylation and activation of tyrosine hydroxylase. Many of the CaMkinases are activated by phosphorylation in addition to binding to CaM.The kinase may autophosphorylate itself, or be phosphorylated by anotherkinase as part of a “kinase cascade”.

Another ligand-activated protein kinase is 5′-AMP-activated proteinkinase (AMPK) (Gao, G. et al. (1996) J. Biol Chem. 15:8675-81).Mammalian AMPK is a regulator of fatty acid and sterol synthesis throughphosphorylation of the enzymes acetyl-CoA carboxylase andhydroxymethylglutaryl-CoA reductase and mediates responses of thesepathways to cellular stresses such as heat shock and depletion ofglucose and ATP. AMPK is a heterotrimeric complex comprised of acatalytic alpha subunit and two non-catalytic beta and gamma subunitsthat are believed to regulate the activity of the alpha subunit.Subunits of AMPK have a much wider distribution in non-lipogenic tissuessuch as brain, heart, spleen, and lung than expected. This distributionsuggests that its role may extend beyond regulation of lipid metabolismalone.

The mitogen-activated protein kinases (MAP) are also members of the STKfamily. MAP kinases also regulate intracellular signaling pathways. Theymediate signal transduction from the cell surface to the nucleus viaphosphorylation cascades. Several subgroups have been identified, andeach manifests different substrate specificities and responds todistinct extracellular stimuli (Egan, S. E. and Weinberg, R. A. (1993)Nature 365:781-783). MAP kinase signaling pathways are present inmammalian cells as well as in yeast. The extracellular stimuli thatactivate mammalian pathways include epidermal growth factor (EGF),ultraviolet light, hyperosmolar medium, heat shock, endotoxiclipopolysaccharide (LPS), and pro-inflammatory cytokines such as tumornecrosis factor (TNF) and interleukin-1 (IL-1).

PRK (proliferation-related kinase) is a serum/cytokine inducible STKthat is involved in regulation of the cell cycle and cell proliferationin human megakaroytic cells (Li, B. et al. (1996) J. Biol. Chem.271:19402-8). PRK is related to the polo (derived from humans polo gene)family of STKs implicated in cell division. PRK is downregulated in lungtumor tissue and may be a proto-oncogene whose deregulated expression innormal tissue leads to oncogenic transformation. Altered MAP kinaseexpression is implicated in a variety of disease conditions includingcancer, inflammation, immune disorders, and disorders affecting growthand development.

The cyclin-dependent protein kinases (CDKs) are another group of STKsthat control the progression of cells through the cell cycle. Cyclinsare small regulatory proteins that act by binding to and activating CDKsthat then trigger various phases of the cell cycle by phosphorylatingand activating selected proteins involved in the mitotic process. CDKsare unique in that they require multiple inputs to become activated. Inaddition to the binding of cyclin, CDK activation requires thephosphorylation of a specific threonine residue and thedephosphorylation of a specific tyrosine residue.

Protein tyrosine kinases, PTKs, specifically phosphorylate tyrosineresidues on their target proteins and may be divided into transmembrane,receptor PTKs and nontransmembrane, non-receptor PTKs. Transmembraneprotein-tyrosine kinases are receptors for most growth factors. Bindingof growth factor to the receptor activates the transfer of a phosphategroup from ATP to selected tyrosine side chains of the receptor andother specific proteins. Growth factors (GF) associated with receptorPTKs include; epidermal GF, platelet-derived GF, fibroblast GF,hepatocyte GF, insulin and insulin-like GFs, nerve GF, vascularendothelial GF, and macrophage colony stimulating factor.

Non-receptor PTKs lack transmembrane regions and, instead, formcomplexes with the intracellular regions of cell surface receptors. Suchreceptors that function through non-receptor PTKs include those forcytokines, hormones (growth hormone and prolactin) and antigen-specificreceptors on T and B lymphocytes.

Many of these PTKs were first identified as the products of mutantoncogenes in cancer cells where their activation was no longer subjectto normal cellular controls. In fact, about one third of the knownoncogenes encode PTKs, and it is well known that cellular transformation(oncogenesis) is often accompanied by increased tyrosine phosphorylationactivity (Carbonneau H and Tonks NK (1992) Annu. Rev. Cell. Biol.8:463-93). Regulation of PTK activity may therefore be an importantstrategy in controlling some types of cancer.

Citron Kinases

The novel human protein, and encoding gene, provided by the presentinvention is related to the serine/threonine kinase family in generaland the subfamily of citron kinases (also referred to asRho-associated-, Rho-binding-, or Rho-interacting-kinases) inparticular. Furthermore, the protein of the present invention is a novelalternative splice form of a protein/gene provided by Applicants in U.S.application Ser. No. 09/804,471, filed Mar. 13, 2001.

Rho GTPases initiate specific kinase cascades upon activation. Forexample, the kinase activity of Rho-binding serine/threonine kinase(ROCK) is increased upon binding to Rho. The citron molecule (Madaule etal., 1995) interacts with Rho and Rac and shares significant structuralhomology with ROCK. Di Cunto et al. (1998) identified a novelserine/threonine kinase, CRIK (citron Rho-interacting kinase), in amouse primary keratinocyte cDNA library. CRIK is a member of themyotonic dystrophy kinase family. 2 different CRIK isoforms have beenfound: a long, 240-kD form of CRIK in which the kinase domain isfollowed by the sequence of citron, and a short, 54-kD form known asCRIK-SK (short kinase), which consists primarily of the kinase domain.CRIK and CRIK-SK proteins are both capable of phosphorylating exogenoussubstrates as well as of autophosphorylation. CRIK kinase activity isstimulated by constitutively active Rho. In keratinocytes, full-lengthCRIK moves into corpuscular cytoplasmic structures where it initiatesrecruitment of actin into these structures. CRIK is expressed inkeratinocytes, brain, spleen, lung, kidney, and highly expressed intestis; Rho-associated kinases ROCK1 and ROCK2 are ubiquitouslyexpressed. CRIK contains a kinase domain, a coiled-coil domain, aleucine-rich domain, a Rho-Rac binding domain, a zinc finger region, apleckstrin homology domain, and a putative SH3-binding domain. Di Cuntoet al. (1998) cloned a human homolog of CRIK and mapped the gene tohuman chromosome 12q.

Di Cunto et al. (2000) used targeted disruption in mice to generate micelacking citron kinase (“citron-K −/− mice”). It was observed that thesecitron-K −/− mice grow at slower rates, are severely ataxic, and die ofseizures before adulthood. The brains of citron-K −/− mice showdefective neurogenesis with dramatic depletion of microneurons in theolfactory bulb, hippocampus, and cerebellum. It was found that theseabnormalities are caused by altered cytokinesis and extreme apoptosisduring development of the central nervous system. Di Cunto et al. (2000)concluded that citron-K is critical for in vivo cytokinesis in neuronalprecursor cells. For a further review of citron kinases, see Di Cunto etal., J Biol Chem Nov. 6, 1998; 273(45):29706-11; Di Cunto, et al.,Neuron 28: 115-127, 2000; Madaule et al., FEBS Lett. 377: 243-248, 1995;and Nagase et al., DNA Res. 6: 63-70, 1999.

Kinase proteins, particularly members of the citron kinase subfamily,are a major target for drug action and development. Accordingly, it isvaluable to the field of pharmaceutical development to identify andcharacterize previously unknown members of this subfamily of kinaseproteins. The present invention advances the state of the art byproviding previously unidentified human kinase proteins that havehomology to members of the citron kinase subfamily.

SUMMARY OF THE INVENTION

The present invention is based in part on the identification of aminoacid sequences of human kinase peptides and proteins that are related tothe citron kinase subfamily, as well as allelic variants and othermammalian orthologs thereof. These unique peptide sequences, and nucleicacid sequences that encode these peptides, can be used as models for thedevelopment of human therapeutic targets, aid in the identification oftherapeutic proteins, and serve as targets for the development of humantherapeutic agents that modulate kinase activity in cells and tissuesthat express the kinase. Experimental data as provided in FIG. 1indicates expression in liver, proliferating human erythroid cells ofthe blood, and glioblastomas of the brain.

DESCRIPTION OF THE FIGURE SHEETS

FIGS. 1A-1B provide the nucleotide sequence of a cDNA molecule thatencodes the kinase protein of the present invention. (SEQ ID NO:1) Inaddition, structure and functional information is provided, such as ATGstart, stop and tissue distribution, where available, that allows one toreadily determine specific uses of inventions based on this molecularsequence. Experimental data as provided in FIG. 1 indicates expressionin liver, proliferating human erythroid cells of the blood, andglioblastomas of the brain.

FIGS. 2A-2F provide the predicted amino acid sequence of the kinase ofthe present invention. (SEQ ID NO:2) In addition structure andfunctional information such as protein family, function, andmodification sites is provided where available, allowing one to readilydetermine specific uses of inventions based on this molecular sequence.

FIGS. 3A-3Z provide genomic sequences that span the gene encoding thekinase protein of the present invention. (SEQ ID NO:3) In additionstructure and functional information, such as intron/exon structure,promoter location, etc., is provided where available, allowing one toreadily determine specific uses of inventions based on this molecularsequence. As illustrated in FIG. 3, SNPs were identified at 13 differentnucleotide positions.

DETAILED DESCRIPTION OF THE INVENTION

General Description

The present invention is based on the sequencing of the human genome.During the sequencing and assembly of the human genome, analysis of thesequence information revealed previously unidentified fragments of thehuman genome that encode peptides that share structural and/or sequencehomology to protein/peptide/domains identified and characterized withinthe art as being a kinase protein or part of a kinase protein and arerelated to the citron kinase subfamily. Utilizing these sequences,additional genomic sequences were assembled and transcript and/or cDNAsequences were isolated and characterized. Based on this analysis, thepresent invention provides amino acid sequences of human kinase peptidesand proteins that are related to the citron kinase subfamily, nucleicacid sequences in the form of transcript sequences, cDNA sequencesand/or genomic sequences that encode these kinase peptides and proteins,nucleic acid variation (allelic information), tissue distribution ofexpression, and information about the closest art knownprotein/peptide/domain that has structural or sequence homology to thekinase of the present invention.

In addition to being previously unknown, the peptides that are providedin the present invention are selected based on their ability to be usedfor the development of commercially important products and services.Specifically, the present peptides are selected based on homology and/orstructural relatedness to known kinase proteins of the citron kinasesubfamily and the expression pattern observed. Experimental data asprovided in FIG. 1 indicates expression in liver, proliferating humanerythroid cells of the blood, and glioblastomas of the brain. The arthas clearly established the commercial importance of members of thisfamily of proteins and proteins that have expression patterns similar tothat of the present gene. Some of the more specific features of thepeptides of the present invention, and the uses thereof, are describedherein, particularly in the Background of the Invention and in theannotation provided in the Figures, and/or are known within the art foreach of the known citron family or subfamily of kinase proteins.

Specific Embodiments

Peptide Molecules

The present invention provides nucleic acid sequences that encodeprotein molecules that have been identified as being members of thekinase family of proteins and are related to the citron kinase subfamily(protein sequences are provided in FIG. 2, transcript/cDNA sequences areprovided in FIG. 1 and genomic sequences are provided in FIG. 3). Thepeptide sequences provided in FIG. 2, as well as the obvious variantsdescribed herein, particularly allelic variants as identified herein andusing the information in FIG. 3, will be referred herein as the kinasepeptides of the present invention, kinase peptides, or peptides/proteinsof the present invention.

The present invention provides isolated peptide and protein moleculesthat consist of, consist essentially of, or comprise the amino acidsequences of the kinase peptides disclosed in the FIG. 2, (encoded bythe nucleic acid molecule shown in FIG. 1, transcript/cDNA or FIG. 3,genomic sequence), as well as all obvious variants of these peptidesthat are within the art to make and use. Some of these variants aredescribed in detail below.

As used herein, a peptide is said to be “isolated” or “purified” when itis substantially free of cellular material or free of chemicalprecursors or other chemicals. The peptides of the present invention canbe purified to homogeneity or other degrees of purity. The level ofpurification will be based on the intended use. The critical feature isthat the preparation allows for the desired function of the peptide,even if in the presence of considerable amounts of other components (thefeatures of an isolated nucleic acid molecule is discussed below).

In some uses, “substantially free of cellular material” includespreparations of the peptide having less than about 30% (by dry weight)other proteins (i.e., contaminating protein), less than about 20% otherproteins, less than about 10% other proteins, or less than about 5%other proteins. When the peptide is recombinantly produced, it can alsobe substantially free of culture medium, i.e., culture medium representsless than about 20% of the volume of the protein preparation.

The language “substantially free of chemical precursors or otherchemicals” includes preparations of the peptide in which it is separatedfrom chemical precursors or other chemicals that are involved in itssynthesis. In one embodiment, the language “substantially free ofchemical precursors or other chemicals” includes preparations of thekinase peptide having less than about 30% (by dry weight) chemicalprecursors or other chemicals, less than about 20% chemical precursorsor other chemicals, less than about 10% chemical precursors or otherchemicals, or less than about 5% chemical precursors or other chemicals.

The isolated kinase peptide can be purified from cells that naturallyexpress it, purified from cells that have been altered to express it(recombinant), or synthesized using known protein synthesis methods.Experimental data as provided in FIG. 1 indicates expression in liver,proliferating human erythroid cells of the blood, and glioblastomas ofthe brain. For example, a nucleic acid molecule encoding the kinasepeptide is cloned into an expression vector, the expression vectorintroduced into a host cell and the protein expressed in the host cell.The protein can then be isolated from the cells by an appropriatepurification scheme using standard protein purification techniques. Manyof these techniques are described in detail below.

Accordingly, the present invention provides proteins that consist of theamino acid sequences provided in FIG. 2 (SEQ ID NO:2), for example,proteins encoded by the transcript/cDNA nucleic acid sequences shown inFIG. 1 (SEQ ID NO:1) and the genomic sequences provided in FIG. 3 (SEQID NO:3). The amino acid sequence of such a protein is provided in FIG.2. A protein consists of an amino acid sequence when the amino acidsequence is the final amino acid sequence of the protein.

The present invention further provides proteins that consist essentiallyof the amino acid sequences provided in FIG. 2 (SEQ ID NO:2), forexample, proteins encoded by the transcript/cDNA nucleic acid sequencesshown in FIG. 1 (SEQ ID NO:1) and the genomic sequences provided in FIG.3 (SEQ ID NO:3). A protein consists essentially of an amino acidsequence when such an amino acid sequence is present with only a fewadditional amino acid residues, for example from about 1 to about 100 orso additional residues, typically from 1 to about 20 additional residuesin the final protein.

The present invention further provides proteins that comprise the aminoacid sequences provided in FIG. 2 (SEQ ID NO:2), for example, proteinsencoded by the transcript/cDNA nucleic acid sequences shown in FIG. 1(SEQ ID NO:1) and the genomic sequences provided in FIG. 3 (SEQ IDNO:3). A protein comprises an amino acid sequence when the amino acidsequence is at least part of the final amino acid sequence of theprotein. In such a fashion, the protein can be only the peptide or haveadditional amino acid molecules, such as amino acid residues (contiguousencoded sequence) that are naturally associated with it or heterologousamino acid residues/peptide sequences. Such a protein can have a fewadditional amino acid residues or can comprise several hundred or moreadditional amino acids. The preferred classes of proteins that arecomprised of the kinase peptides of the present invention are thenaturally occurring mature proteins. A brief description of how varioustypes of these proteins can be made/isolated is provided below.

The kinase peptides of the present invention can be attached toheterologous sequences to form chimeric or fusion proteins. Suchchimeric and fusion proteins comprise a kinase peptide operativelylinked to a heterologous protein having an amino acid sequence notsubstantially homologous to the kinase peptide. “Operatively linked”indicates that the kinase peptide and the heterologous protein are fusedin-frame. The heterologous protein can be fused to the N-terminus orC-terminus of the kinase peptide.

In some uses, the fusion protein does not affect the activity of thekinase peptide per se. For example, the fusion protein can include, butis not limited to, enzymatic fusion proteins, for examplebeta-galactosidase fusions, yeast two-hybrid GAL fusions, poly-Hisfusions, MYC-tagged, HI-tagged and Ig fusions. Such fusion proteins,particularly poly-His fusions, can facilitate the purification ofrecombinant kinase peptide. In certain host cells (e.g., mammalian hostcells), expression and/or secretion of a protein can be increased byusing a heterologous signal sequence.

A chimeric or fusion protein can be produced by standard recombinant DNAtechniques.

For example, DNA fragments coding for the different protein sequencesare ligated together in-frame in accordance with conventionaltechniques. In another embodiment, the fusion gene can be synthesized byconventional techniques including automated DNA synthesizers.Alternatively, PCR amplification of gene fragments can be carried outusing anchor primers which give rise to complementary overhangs betweentwo consecutive gene fragments which can subsequently be annealed andre-amplified to generate a chimeric gene sequence (see Ausubel et al.,Current Protocols in Molecular Biology, 1992). Moreover, many expressionvectors are commercially available that already encode a fusion moiety(e.g., a GST protein). A kinase peptide-encoding nucleic acid can becloned into such an expression vector such that the fusion moiety islinked in-frame to the kinase peptide.

As mentioned above, the present invention also provides and enablesobvious variants of the amino acid sequence of the proteins of thepresent invention, such as naturally occurring mature forms of thepeptide, allelic/sequence variants of the peptides, non-naturallyoccurring recombinantly derived variants of the peptides, and orthologsand paralogs of the peptides. Such variants can readily be generatedusing art-known techniques in the fields of recombinant nucleic acidtechnology and protein biochemistry. It is understood, however, thatvariants exclude any amino acid sequences disclosed prior to theinvention.

Such variants can readily be identified/made using molecular techniquesand the sequence information disclosed herein. Further, such variantscan readily be distinguished from other peptides based on sequenceand/or structural homology to the kinase peptides of the presentinvention. The degree of homology/identity present will be basedprimarily on whether the peptide is a functional variant ornon-functional variant, the amount of divergence present in the paralogfamily and the evolutionary distance between the orthologs.

To determine the percent identity of two amino acid sequences or twonucleic acid sequences, the sequences are aligned for optimal comparisonpurposes (e.g., gaps can be introduced in one or both of a first and asecond amino acid or nucleic acid sequence for optimal alignment andnon-homologous sequences can be disregarded for comparison purposes). Ina preferred embodiment, at least 30%, 40%, 50%, 60%, 70%, 80%, or 90% ormore of the length of a reference sequence is aligned for comparisonpurposes. The amino acid residues or nucleotides at corresponding aminoacid positions or nucleotide positions are then compared. When aposition in the first sequence is occupied by the same amino acidresidue or nucleotide as the corresponding position in the secondsequence, then the molecules are identical at that position (as usedherein amino acid or nucleic acid “identity” is equivalent to amino acidor nucleic acid “homology”). The percent identity between the twosequences is a function of the number of identical positions shared bythe sequences, taking into account the number of gaps, and the length ofeach gap, which need to be introduced for optimal alignment of the twosequences.

The comparison of sequences and determination of percent identity andsimilarity between two sequences can be accomplished using amathematical algorithm. (Computational Molecular Biology, Lesk, A. M.,ed., Oxford University Press, New York, 1988; Biocomputing: Informaticsand Genome Projects, Smith, D. W., ed., Academic Press, New York, 1993;Computer Analysis of Sequence Data, Part 1, Griffin, A. M., and Griffin,H. G., eds., Humana Press, New Jersey, 1994; Sequence Analysis inMolecular Biology, von Heinje, G., Academic Press, 1987; and SequenceAnalysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press,New York, 1991). In a preferred embodiment, the percent identity betweentwo amino acid sequences is determined using the Needleman and Wunsch(J. Mol. Biol. (48):444-453 (1970)) algorithm which has beenincorporated into the GAP program in the GCG software package (availableat http://www.gcg.com), using either a Blossom 62 matrix or a PAM250matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a lengthweight of 1, 2, 3, 4, 5, or 6. In yet another preferred embodiment, thepercent identity between two nucleotide sequences is determined usingthe GAP program in the GCG software package (Devereux, J., et al.,Nucleic Acids Res. 12(1):387 (1984)) (available at http://www.gcg.com),using a NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80and a length weight of 1, 2, 3, 4, 5, or 6. In another embodiment, thepercent identity between two amino acid or nucleotide sequences isdetermined using the algorithm of E. Myers and W. Miller (CABIOS,4:11-17 (1989)) which has been incorporated into the ALIGN program(version 2.0), using a PAM120 weight residue table, a gap length penaltyof 12 and a gap penalty of 4.

The nucleic acid and protein sequences of the present invention canfurther be used as a “query sequence” to perform a search againstsequence databases to, for example, identify other family members orrelated sequences. Such searches can be performed using the NBLAST andXBLAST programs (version 2.0) of Altschul, et al. (J. Mol. Biol.215:403-10 (1990)). BLAST nucleotide searches can be performed with theNBLAST program, score=100, wordlength=12 to obtain nucleotide sequenceshomologous to the nucleic acid molecules of the invention. BLAST proteinsearches can be performed with the XBLAST program, score=50,wordlength=3 to obtain amino acid sequences homologous to the proteinsof the invention. To obtain gapped alignments for comparison purposes,Gapped BLAST can be utilized as described in Altschul et al. (NucleicAcids Res. 25(17):3389-3402 (1997)). When utilizing BLAST and gappedBLAST programs, the default parameters of the respective programs (e.g.,XBLAST and NBLAST) can be used.

Full-length pre-processed forms, as well as mature processed forms, ofproteins that comprise one of the peptides of the present invention canreadily be identified as having complete sequence identity to one of thekinase peptides of the present invention as well as being encoded by thesame genetic locus as the kinase peptide provided herein. As indicatedin FIG. 3, the map position of the kinase gene of the present inventionwas determined to be on human chromosome 12.

Allelic variants of a kinase peptide can readily be identified as beinga human protein having a high degree (significant) of sequencehomology/identity to at least a portion of the kinase peptide as well asbeing encoded by the same genetic locus as the kinase peptide providedherein. Genetic locus can readily be determined based on the genomicinformation provided in FIG. 3, such as the genomic sequence mapped tothe reference human. As indicated in FIG. 3, the map position of thekinase gene of the present invention was determined to be on humanchromosome 12. As used herein, two proteins (or a region of theproteins) have significant homology when the amino acid sequences aretypically at least about 70-80%, 80-90%, and more typically at leastabout 90-95% or more homologous. A significantly homologous amino acidsequence, according to the present invention, will be encoded by anucleic acid sequence that will hybridize to a kinase peptide encodingnucleic acid molecule under stringent conditions as more fully describedbelow.

FIG. 3 provides information on SNPs that have been identified at 13different nucleotide positions in the gene encoding the kinase proteinsof the present invention.

Paralogs of a kinase peptide can readily be identified as having somedegree of significant sequence homology/identity to at least a portionof the kinase peptide, as being encoded by a gene from humans, and ashaving similar activity or function. Two proteins will typically beconsidered paralogs when the amino acid sequences are typically at leastabout 60% or greater, and more typically at least about 70% or greaterhomology through a given region or domain. Such paralogs will be encodedby a nucleic acid sequence that will hybridize to a kinase peptideencoding nucleic acid molecule under moderate to stringent conditions asmore fully described below.

Orthologs of a kinase peptide can readily be identified as having somedegree of significant sequence homology/identity to at least a portionof the kinase peptide as well as being encoded by a gene from anotherorganism. Preferred orthologs will be isolated from mammals, preferablyprimates, for the development of human therapeutic targets and agents.Such orthologs will be encoded by a nucleic acid sequence that willhybridize to a kinase peptide encoding nucleic acid molecule undermoderate to stringent conditions, as more fully described below,depending on the degree of relatedness of the two organisms yielding theproteins.

Non-naturally occurring variants of the kinase peptides of the presentinvention can readily be generated using recombinant techniques. Suchvariants include, but are not limited to deletions, additions andsubstitutions in the amino acid sequence of the kinase peptide. Forexample, one class of substitutions are conserved amino acidsubstitution. Such substitutions are those that substitute a given aminoacid in a kinase peptide by another amino acid of like characteristics.Typically seen as conservative substitutions are the replacements, onefor another, among the aliphatic amino acids Ala, Val, Leu, and Ile;interchange of the hydroxyl residues Ser and Thr; exchange of the acidicresidues Asp and Glu; substitution between the amide residues Asn andGln; exchange of the basic residues Lys and Arg; and replacements amongthe aromatic residues Phe and Tyr. Guidance concerning which amino acidchanges are likely to be phenotypically silent are found in Bowie etal., Science 247:1306-1310 (1990).

Variant kinase peptides can be fully functional or can lack function inone or more activities, e.g. ability to bind substrate, ability tophosphorylate substrate, ability to mediate signaling, etc. Fullyfunctional variants typically contain only conservative variation orvariation in non-critical residues or in non-critical regions. FIG. 2provides the result of protein analysis and can be used to identifycritical domains/regions. Functional variants can also containsubstitution of similar amino acids that result in no change or aninsignificant change in function. Alternatively, such substitutions maypositively or negatively affect function to some degree.

Non-functional variants typically contain one or more non-conservativeamino acid substitutions, deletions, insertions, inversions, ortruncation or a substitution, insertion, inversion, or deletion in acritical residue or critical region.

Amino acids that are essential for function can be identified by methodsknown in the art, such as site-directed mutagenesis or alanine-scanningmutagenesis (Cunningham et al., Science 244:1081-1085 (1989)),particularly using the results provided in FIG. 2. The latter procedureintroduces single alanine mutations at every residue in the molecule.The resulting mutant molecules are then tested for biological activitysuch as kinase activity or in assays such as an in vitro proliferativeactivity. Sites that are critical for binding partner/substrate bindingcan also be determined by structural analysis such as crystallization,nuclear magnetic resonance or photoaffinity labeling (Smith et al., J.Mol. Biol. 224:899-904 (1992); de Vos et al. Science 255:306-312(1992)).

The present invention further provides fragments of the kinase peptides,in addition to proteins and peptides that comprise and consist of suchfragments, particularly those comprising the residues identified in FIG.2. The fragments to which the invention pertains, however, are not to beconstrued as encompassing fragments that may be disclosed publicly priorto the present invention.

As used herein, a fragment comprises at least 8, 10, 12, 14, 16, or morecontiguous amino acid residues from a kinase peptide. Such fragments canbe chosen based on the ability to retain one or more of the biologicalactivities of the kinase peptide or could be chosen for the ability toperform a function, e.g. bind a substrate or act as an immunogen.Particularly important fragments are biologically active fragments,peptides that are, for example, about 8 or more amino acids in length.Such fragments will typically comprise a domain or motif of the kinasepeptide, e.g., active site, a transmembrane domain or asubstrate-binding domain. Further, possible fragments include, but arenot limited to, domain or motif containing fragments, soluble peptidefragments, and fragments containing immunogenic structures. Predicteddomains and functional sites are readily identifiable by computerprograms well known and readily available to those of skill in the art(e.g., PROSITE analysis). The results of one such analysis are providedin FIG. 2.

Polypeptides often contain amino acids other than the 20 amino acidscommonly referred to as the 20 naturally occurring amino acids. Further,many amino acids, including the terminal amino acids, may be modified bynatural processes, such as processing and other post-translationalmodifications, or by chemical modification techniques well known in theart. Common modifications that occur naturally in kinase peptides aredescribed in basic texts, detailed monographs, and the researchliterature, and they are well known to those of skill in the art (someof these features are identified in FIG. 2).

Known modifications include, but are not limited to, acetylation,acylation, ADP-ribosylation, amidation, covalent attachment of flavin,covalent attachment of a heme moiety, covalent attachment of anucleotide or nucleotide derivative, covalent attachment of a lipid orlipid derivative, covalent attachment of phosphotidylinositol,cross-linking, cyclization, disulfide bond formation, demethylation,formation of covalent crosslinks, formation of cystine, formation ofpyroglutamate, formylation, gamma carboxylation, glycosylation, GPIanchor formation, hydroxylation, iodination, methylation,myristoylation, oxidation, proteolytic processing, phosphorylation,prenylation, racemization, selenoylation, sulfation, transfer-RNAmediated addition of amino acids to proteins such as arginylation, andubiquitination.

Such modifications are well known to those of skill in the art and havebeen described in great detail in the scientific literature. Severalparticularly common modifications, glycosylation, lipid attachment,sulfation, gamma-carboxylation of glutamic acid residues, hydroxylationand ADP-ribosylation, for instance, are described in most basic texts,such as Proteins—Structure and Molecular Properties, 2nd Ed., T. E.Creighton, W. H. Freeman and Company, New York (1993). Many detailedreviews are available on this subject, such as by Wold, F.,Posttranslational Covalent Modification of Proteins, B. C. Johnson, Ed.,Academic Press, New York 1-12 (1983); Seifter et al. (Meth. Enzymol.182: 626-646 (1990)) and Rattan et al. (Ann. N.Y. Acad Sci. 663:48-62(1992)).

Accordingly, the kinase peptides of the present invention also encompassderivatives or analogs in which a substituted amino acid residue is notone encoded by the genetic code, in which a substituent group isincluded, in which the mature kinase peptide is fused with anothercompound, such as a compound to increase the half-life of the kinasepeptide (for example, polyethylene glycol), or in which the additionalamino acids are fused to the mature kinase peptide, such as a leader orsecretory sequence or a sequence for purification of the mature kinasepeptide or a pro-protein sequence.

Protein/Peptide Uses

The proteins of the present invention can be used in substantial andspecific assays related to the functional information provided in theFigures; to raise antibodies or to elicit another immune response; as areagent (including the labeled reagent) in assays designed toquantitatively determine levels of the protein (or its binding partneror ligand) in biological fluids; and as markers for tissues in which thecorresponding protein is preferentially expressed (either constitutivelyor at a particular stage of tissue differentiation or development or ina disease state). Where the protein binds or potentially binds toanother protein or ligand (such as, for example, in a kinase-effectorprotein interaction or kinase-ligand interaction), the protein can beused to identify the binding partner/ligand so as to develop a system toidentify inhibitors of the binding interaction. Any or all of these usesare capable of being developed into reagent grade or kit format forcommercialization as commercial products.

Methods for performing the uses listed above are well known to thoseskilled in the art. References disclosing such methods include“Molecular Cloning: A Laboratory Manual”, 2d ed., Cold Spring HarborLaboratory Press, Sambrook, J., E. F. Fritsch and T. Maniatis eds.,1989, and “Methods in Enzymology: Guide to Molecular CloningTechniques”, Academic Press, Berger, S. L. and A. R. Kimmel eds., 1987.

The potential uses of the peptides of the present invention are basedprimarily on the source of the protein as well as the class/action ofthe protein. For example, kinases isolated from humans and theirhuman/mammalian orthologs serve as targets for identifying agents foruse in mammalian therapeutic applications, e.g. a human drug,particularly in modulating a biological or pathological response in acell or tissue that expresses the kinase. Experimental data as providedin FIG. 1 indicates that kinase proteins of the present invention areexpressed in proliferating human erythroid cells of the blood and inglioblastomas of the brain, as indicated by virtual northern blotanalysis. Additionally, the tissue source of the cDNA clone of thepresent invention indicates expression in the liver. A large percentageof pharmaceutical agents are being developed that modulate the activityof kinase proteins, particularly members of the citron subfamily (seeBackground of the Invention). The structural and functional informationprovided in the Background and Figures provide specific and substantialuses for the molecules of the present invention, particularly incombination with the expression information provided in FIG. 1.Experimental data as provided in FIG. 1 indicates expression in liver,proliferating human erythroid cells of the blood, and glioblastomas ofthe brain. Such uses can readily be determined using the informationprovided herein, that which is known in the art, and routineexperimentation.

The proteins of the present invention (including variants and fragmentsthat may have been disclosed prior to the present invention) are usefulfor biological assays related to kinases that are related to members ofthe citron subfamily. Such assays involve any of the known kinasefunctions or activities or properties useful for diagnosis and treatmentof kinase-related conditions that are specific for the subfamily ofkinases that the one of the present invention belongs to, particularlyin cells and tissues that express the kinase. Experimental data asprovided in FIG. 1 indicates that kinase proteins of the presentinvention are expressed in proliferating human erythroid cells of theblood and in glioblastomas of the brain, as indicated by virtualnorthern blot analysis. Additionally, the tissue source of the cDNAclone of the present invention indicates expression in the liver.

The proteins of the present invention are also useful in drug screeningassays, in cell-based or cell-free systems. Cell-based systems can benative, i.e., cells that normally express the kinase, as a biopsy orexpanded in cell culture. Experimental data as provided in FIG. 1indicates expression in liver, proliferating human erythroid cells ofthe blood, and glioblastomas of the brain. In an alternate embodiment,cell-based assays involve recombinant host cells expressing the kinaseprotein.

The polypeptides can be used to identify compounds that modulate kinaseactivity of the protein in its natural state or an altered form thatcauses a specific disease or pathology associated with the kinase. Boththe kinases of the present invention and appropriate variants andfragments can be used in high-throughput screens to assay candidatecompounds for the ability to bind to the kinase. These compounds can befurther screened against a functional kinase to determine the effect ofthe compound on the kinase activity. Further, these compounds can betested in animal or invertebrate systems to determineactivity/effectiveness. Compounds can be identified that activate(agonist) or inactivate (antagonist) the kinase to a desired degree.

Further, the proteins of the present invention can be used to screen acompound for the ability to stimulate or inhibit interaction between thekinase protein and a molecule that normally interacts with the kinaseprotein, e.g. a substrate or a component of the signal pathway that thekinase protein normally interacts (for example, another kinase). Suchassays typically include the steps of combining the kinase protein witha candidate compound under conditions that allow the kinase protein, orfragment, to interact with the target molecule, and to detect theformation of a complex between the protein and the target or to detectthe biochemical consequence of the interaction with the kinase proteinand the target, such as any of the associated effects of signaltransduction such as protein phosphorylation, cAMP turnover, andadenylate cyclase activation, etc.

Candidate compounds include, for example, 1) peptides such as solublepeptides, including Ig-tailed fusion peptides and members of randompeptide libraries (see, e.g., Lam et al., Nature 354:82-84 (1991);Houghten et al., Nature 354:84-86 (1991)) and combinatorialchemistry-derived molecular libraries made of D- and/or L-configurationamino acids; 2) phosphopeptides (e.g., members of random and partiallydegenerate, directed phosphopeptide libraries, see, e.g., Songyang etal., Cell 72:767-778 (1993)); 3) antibodies (e.g., polyclonal,monoclonal, humanized, anti-idiotypic, chimeric, and single chainantibodies as well as Fab, F(ab′)₂, Fab expression library fragments,and epitope-binding fragments of antibodies); and 4) small organic andinorganic molecules (e.g., molecules obtained from combinatorial andnatural product libraries).

One candidate compound is a soluble fragment of the receptor thatcompetes for substrate binding. Other candidate compounds include mutantkinases or appropriate fragments containing mutations that affect kinasefunction and thus compete for substrate. Accordingly, a fragment thatcompetes for substrate, for example with a higher affinity, or afragment that binds substrate but does not allow release, is encompassedby the invention.

The invention further includes other end point assays to identifycompounds that modulate (stimulate or inhibit) kinase activity. Theassays typically involve an assay of events in the signal transductionpathway that indicate kinase activity. Thus, the phosphorylation of asubstrate, activation of a protein, a change in the expression of genesthat are up- or down-regulated in response to the kinase proteindependent signal cascade can be assayed.

Any of the biological or biochemical functions mediated by the kinasecan be used as an endpoint assay. These include all of the biochemicalor biochemical/biological events described herein, in the referencescited herein, incorporated by reference for these endpoint assaytargets, and other functions known to those of ordinary skill in the artor that can be readily identified using the information provided in theFigures, particularly FIG. 2. Specifically, a biological function of acell or tissues that expresses the kinase can be assayed. Experimentaldata as provided in FIG. 1 indicates that kinase proteins of the presentinvention are expressed in proliferating human erythroid cells of theblood and in glioblastomas of the brain, as indicated by virtualnorthern blot analysis. Additionally, the tissue source of the cDNAclone of the present invention indicates expression in the liver.

Binding and/or activating compounds can also be screened by usingchimeric kinase proteins in which the amino terminal extracellulardomain, or parts thereof, the entire transmembrane domain or subregions,such as any of the seven transmembrane segments or any of theintracellular or extracellular loops and the carboxy terminalintracellular domain, or parts thereof, can be replaced by heterologousdomains or subregions. For example, a substrate-binding region can beused that interacts with a different substrate then that which isrecognized by the native kinase. Accordingly, a different set of signaltransduction components is available as an end-point assay foractivation. This allows for assays to be performed in other than thespecific host cell from which the kinase is derived.

The proteins of the present invention are also useful in competitionbinding assays in methods designed to discover compounds that interactwith the kinase (e.g. binding partners and/or ligands). Thus, a compoundis exposed to a kinase polypeptide under conditions that allow thecompound to bind or to otherwise interact with the polypeptide. Solublekinase polypeptide is also added to the mixture. If the test compoundinteracts with the soluble kinase polypeptide, it decreases the amountof complex formed or activity from the kinase target. This type of assayis particularly useful in cases in which compounds are sought thatinteract with specific regions of the kinase. Thus, the solublepolypeptide that competes with the target kinase region is designed tocontain peptide sequences corresponding to the region of interest.

To perform cell free drug screening assays, it is sometimes desirable toimmobilize either the kinase protein, or fragment, or its targetmolecule to facilitate separation of complexes from uncomplexed forms ofone or both of the proteins, as well as to accommodate automation of theassay.

Techniques for immobilizing proteins on matrices can be used in the drugscreening assays. In one embodiment, a fusion protein can be providedwhich adds a domain that allows the protein to be bound to a matrix. Forexample, glutathione-S-transferase fusion proteins can be adsorbed ontoglutathione sepharose beads (Sigma Chemical, St. Louis, Mo.) orglutathione derivatized microtitre plates, which are then combined withthe cell lysates (e.g., ³⁵S-labeled) and the candidate compound, and themixture incubated under conditions conducive to complex formation (e.g.,at physiological conditions for salt and pH). Following incubation, thebeads are washed to remove any unbound label, and the matrix immobilizedand radiolabel determined directly, or in the supernatant after thecomplexes are dissociated. Alternatively, the complexes can bedissociated from the matrix, separated by SDS-PAGE, and the level ofkinase-binding protein found in the bead fraction quantitated from thegel using standard electrophoretic techniques. For example, either thepolypeptide or its target molecule can be immobilized utilizingconjugation of biotin and streptavidin using techniques well known inthe art. Alternatively, antibodies reactive with the protein but whichdo not interfere with binding of the protein to its target molecule canbe derivatized to the wells of the plate, and the protein trapped in thewells by antibody conjugation. Preparations of a kinase-binding proteinand a candidate compound are incubated in the kinase protein-presentingwells and the amount of complex trapped in the well can be quantitated.Methods for detecting such complexes, in addition to those describedabove for the GST-immobilized complexes, include immunodetection ofcomplexes using antibodies reactive with the kinase protein targetmolecule, or which are reactive with kinase protein and compete with thetarget molecule, as well as enzyme-linked assays which rely on detectingan enzymatic activity associated with the target molecule.

Agents that modulate one of the kinases of the present invention can beidentified using one or more of the above assays, alone or incombination. It is generally preferable to use a cell-based or cell freesystem first and then confirm activity in an animal or other modelsystem. Such model systems are well known in the art and can readily beemployed in this context.

Modulators of kinase protein activity identified according to these drugscreening assays can be used to treat a subject with a disorder mediatedby the kinase pathway, by treating cells or tissues that express thekinase. Experimental data as provided in FIG. 1 indicates expression inliver, proliferating human erythroid cells of the blood, andglioblastomas of the brain. These methods of treatment include the stepsof administering a modulator of kinase activity in a pharmaceuticalcomposition to a subject in need of such treatment, the modulator beingidentified as described herein.

In yet another aspect of the invention, the kinase proteins can be usedas “bait proteins” in a two-hybrid assay or three-hybrid assay (see,e.g., U.S. Pat. No. 5,283,317; Zervos et al. (1993) Cell 72:223-232;Madura et al. (1993) J. Biol. Chem. 268:12046-12054; Bartel et al.(1993) Biotechniques 14:920-924; Iwabuchi et al. (1993) Oncogene8:1693-1696; and Brent WO94/10300), to identify other proteins, whichbind to or interact with the kinase and are involved in kinase activity.Such kinase-binding proteins are also likely to be involved in thepropagation of signals by the kinase proteins or kinase targets as, forexample, downstream elements of a kinase-mediated signaling pathway.Alternatively, such kinase-binding proteins are likely to be kinaseinhibitors.

The two-hybrid system is based on the modular nature of mosttranscription factors, which consist of separable DNA-binding andactivation domains. Briefly, the assay utilizes two different DNAconstructs. In one construct, the gene that codes for a kinase proteinis fused to a gene encoding the DNA binding domain of a knowntranscription factor (e.g., GAL-4). In the other construct, a DNAsequence, from a library of DNA sequences, that encodes an unidentifiedprotein (“prey” or “sample”) is fused to a gene that codes for theactivation domain of the known transcription factor. If the “bait” andthe “prey” proteins are able to interact, in vivo, forming akinase-dependent complex, the DNA-binding and activation domains of thetranscription factor are brought into close proximity. This proximityallows transcription of a reporter gene (e.g., LacZ) which is operablylinked to a transcriptional regulatory site responsive to thetranscription factor. Expression of the reporter gene can be detectedand cell colonies containing the functional transcription factor can beisolated and used to obtain the cloned gene which encodes the proteinwhich interacts with the kinase protein.

This invention further pertains to novel agents identified by theabove-described screening assays. Accordingly, it is within the scope ofthis invention to further use an agent identified as described herein inan appropriate animal model. For example, an agent identified asdescribed herein (e.g., a kinase-modulating agent, an antisense kinasenucleic acid molecule, a kinase-specific antibody, or a kinase-bindingpartner) can be used in an animal or other model to determine theefficacy, toxicity, or side effects of treatment with such an agent.Alternatively, an agent identified as described herein can be used in ananimal or other model to determine the mechanism of action of such anagent. Furthermore, this invention pertains to uses of novel agentsidentified by the above-described screening assays for treatments asdescribed herein.

The kinase proteins of the present invention are also useful to providea target for diagnosing a disease or predisposition to disease mediatedby the peptide. Accordingly, the invention provides methods fordetecting the presence, or levels of, the protein (or encoding mRNA) ina cell, tissue, or organism. Experimental data as provided in FIG. 1indicates expression in liver, proliferating human erythroid cells ofthe blood, and glioblastomas of the brain. The method involvescontacting a biological sample with a compound capable of interactingwith the kinase protein such that the interaction can be detected. Suchan assay can be provided in a single detection format or amulti-detection format such as an antibody chip array.

One agent for detecting a protein in a sample is an antibody capable ofselectively binding to protein. A biological sample includes tissues,cells and biological fluids isolated from a subject, as well as tissues,cells and fluids present within a subject.

The peptides of the present invention also provide targets fordiagnosing active protein activity, disease, or predisposition todisease, in a patient having a variant peptide, particularly activitiesand conditions that are known for other members of the family ofproteins to which the present one belongs. Thus, the peptide can beisolated from a biological sample and assayed for the presence of agenetic mutation that results in aberrant peptide. This includes aminoacid substitution, deletion, insertion, rearrangement, (as the result ofaberrant splicing events), and inappropriate post-translationalmodification. Analytic methods include altered electrophoretic mobility,altered tryptic peptide digest, altered kinase activity in cell-based orcell-free assay, alteration in substrate or antibody-binding pattern,altered isoelectric point, direct amino acid sequencing, and any otherof the known assay techniques useful for detecting mutations in aprotein. Such an assay can be provided in a single detection format or amulti-detection format such as an antibody chip array.

In vitro techniques for detection of peptide include enzyme linkedimmunosorbent assays (ELISAs), Western blots, immunoprecipitations andimmunofluorescence using a detection reagent, such as an antibody orprotein binding agent. Alternatively, the peptide can be detected invivo in a subject by introducing into the subject a labeled anti-peptideantibody or other types of detection agent. For example, the antibodycan be labeled with a radioactive marker whose presence and location ina subject can be detected by standard imaging techniques. Particularlyuseful are methods that detect the allelic variant of a peptideexpressed in a subject and methods which detect fragments of a peptidein a sample.

The peptides are also useful in pharmacogenomic analysis.Pharmacogenomics deal with clinically significant hereditary variationsin the response to drugs due to altered drug disposition and abnormalaction in affected persons. See, e.g., Eichelbaum, M. (Clin. Exp.Pharmacol. Physiol. 23(10-11):983-985 (1996)), and Linder, M. W. (Clin.Chem. 43(2):254-266 (1997) outcomes of these variations result in severetoxicity of therapeutic drugs in certain individuals or therapeuticfailure of drugs in certain individuals as a result of individualvariation in metabolism. Thus, the genotype of the individual candetermine the way a therapeutic compound acts on the body or the way thebody metabolizes the compound. Further, the activity of drugmetabolizing enzymes effects both the intensity and duration of drugaction. Thus, the pharmacogenomics of the individual permit theselection of effective compounds and effective dosages of such compoundsfor prophylactic or therapeutic treatment based on the individual'sgenotype. The discovery of genetic polymorphisms in some drugmetabolizing enzymes has explained why some patients do not obtain theexpected drug effects, show an exaggerated drug effect, or experienceserious toxicity from standard drug dosages. Polymorphisms can beexpressed in the phenotype of the extensive metabolizer and thephenotype of the poor metabolizer. Accordingly, genetic polymorphism maylead to allelic protein variants of the kinase protein in which one ormore of the kinase functions in one population is different from thosein another population. The peptides thus allow a target to ascertain agenetic predisposition that can affect treatment modality. Thus, in aligand-based treatment, polymorphism may give rise to amino terminalextracellular domains and/or other substrate-binding regions that aremore or less active in substrate binding, and kinase activation.Accordingly, substrate dosage would necessarily be modified to maximizethe therapeutic effect within a given population containing apolymorphism. As an alternative to genotyping, specific polymorphicpeptides could be identified.

The peptides are also useful for treating a disorder characterized by anabsence of, inappropriate, or unwanted expression of the protein.Experimental data as provided in FIG. 1 indicates expression in liver,proliferating human erythroid cells of the blood, and glioblastomas ofthe brain. Accordingly, methods for treatment include the use of thekinase protein or fragments.

Antibodies

The invention also provides antibodies that selectively bind to one ofthe peptides of the present invention, a protein comprising such apeptide, as well as variants and fragments thereof. As used herein, anantibody selectively binds a target peptide when it binds the targetpeptide and does not significantly bind to unrelated proteins. Anantibody is still considered to selectively bind a peptide even if italso binds to other proteins that are not substantially homologous withthe target peptide so long as such proteins share homology with afragment or domain of the peptide target of the antibody. In this case,it would be understood that antibody binding to the peptide is stillselective despite some degree of cross-reactivity.

As used herein, an antibody is defined in terms consistent with thatrecognized within the art: they are multi-subunit proteins produced by amammalian organism in response to an antigen challenge. The antibodiesof the present invention include polyclonal antibodies and monoclonalantibodies, as well as fragments of such antibodies, including, but notlimited to, Fab or F(ab′)₂, and Fv fragments.

Many methods are known for generating and/or identifying antibodies to agiven target peptide. Several such methods are described by Harlow,Antibodies, Cold Spring Harbor Press, (1989).

In general, to generate antibodies, an isolated peptide is used as animmunogen and is administered to a mammalian organism, such as a rat,rabbit or mouse. The full-length protein, an antigenic peptide fragmentor a fusion protein can be used. Particularly important fragments arethose covering functional domains, such as the domains identified inFIG. 2, and domain of sequence homology or divergence amongst thefamily, such as those that can readily be identified using proteinalignment methods and as presented in the Figures.

Antibodies are preferably prepared from regions or discrete fragments ofthe kinase proteins. Antibodies can be prepared from any region of thepeptide as described herein. However, preferred regions will includethose involved in function/activity and/or kinase/binding partnerinteraction. FIG. 2 can be used to identify particularly importantregions while sequence alignment can be used to identify conserved andunique sequence fragments.

An antigenic fragment will typically comprise at least 8 contiguousamino acid residues. The antigenic peptide can comprise, however, atleast 10, 12, 14, 16 or more amino acid residues. Such fragments can beselected on a physical property, such as fragments correspond to regionsthat are located on the surface of the protein, e.g., hydrophilicregions or can be selected based on sequence uniqueness (see FIG. 2).

Detection on an antibody of the present invention can be facilitated bycoupling (i.e., physically linking) the antibody to a detectablesubstance. Examples of detectable substances include various enzymes,prosthetic groups, fluorescent materials, luminescent materials,bioluminescent materials, and radioactive materials. Examples ofsuitable enzymes include horseradish peroxidase, alkaline phosphatase,β-galactosidase, or acetylcholinesterase; examples of suitableprosthetic group complexes include streptavidinibiotin andavidin/biotin; examples of suitable fluorescent materials includeumbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine,dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; anexample of a luminescent material includes luminol; examples ofbioluminescent materials include luciferase, luciferin, and aequorin,and examples of suitable radioactive material include ¹²⁵I, ¹³¹I, ³⁵S or³H.

Antibody Uses

The antibodies can be used to isolate one of the proteins of the presentinvention by standard techniques, such as affinity chromatography orimmunoprecipitation. The antibodies can facilitate the purification ofthe natural protein from cells and recombinantly produced proteinexpressed in host cells. In addition, such antibodies are useful todetect the presence of one of the proteins of the present invention incells or tissues to determine the pattern of expression of the proteinamong various tissues in an organism and over the course of normaldevelopment. Experimental data as provided in FIG. 1 indicates thatkinase proteins of the present invention are expressed in proliferatinghuman erythroid cells of the blood and in glioblastomas of the brain, asindicated by virtual northern blot analysis. Additionally, the tissuesource of the cDNA clone of the present invention indicates expressionin the liver. Further, such antibodies can be used to detect protein insitu, in vitro, or in a cell lysate or supernatant in order to evaluatethe abundance and pattern of expression. Also, such antibodies can beused to assess abnormal tissue distribution or abnormal expressionduring development or progression of a biological condition. Antibodydetection of circulating fragments of the full length protein can beused to identify turnover.

Further, the antibodies can be used to assess expression in diseasestates such as in active stages of the disease or in an individual witha predisposition toward disease related to the protein's function. Whena disorder is caused by an inappropriate tissue distribution,developmental expression, level of expression of the protein, orexpressed/processed form, the antibody can be prepared against thenormal protein. Experimental data as provided in FIG. 1 indicatesexpression in liver, proliferating human erythroid cells of the blood,and glioblastomas of the brain. If a disorder is characterized by aspecific mutation in the protein, antibodies specific for this mutantprotein can be used to assay for the presence of the specific mutantprotein.

The antibodies can also be used to assess normal and aberrantsubcellular localization of cells in the various tissues in an organism.Experimental data as provided in FIG. 1 indicates expression in liver,proliferating human erythroid cells of the blood, and glioblastomas ofthe brain. The diagnostic uses can be applied, not only in genetictesting, but also in monitoring a treatment modality. Accordingly, wheretreatment is ultimately aimed at correcting expression level or thepresence of aberrant sequence and aberrant tissue distribution ordevelopmental expression, antibodies directed against the protein orrelevant fragments can be used to monitor therapeutic efficacy.

Additionally, antibodies are useful in pharmacogenomic analysis. Thus,antibodies prepared against polymorphic proteins can be used to identifyindividuals that require modified treatment modalities. The antibodiesare also useful as diagnostic tools as an immunological marker foraberrant protein analyzed by electrophoretic mobility, isoelectricpoint, tryptic peptide digest, and other physical assays known to thosein the art.

The antibodies are also useful for tissue typing. Experimental data asprovided in FIG. 1 indicates expression in liver, proliferating humanerythroid cells of the blood, and glioblastomas of the brain. Thus,where a specific protein has been correlated with expression in aspecific tissue, antibodies that are specific for this protein can beused to identify a tissue type.

The antibodies are also useful for inhibiting protein function, forexample, blocking the binding of the kinase peptide to a binding partnersuch as a substrate. These uses can also be applied in a therapeuticcontext in which treatment involves inhibiting the protein's function.An antibody can be used, for example, to block binding, thus modulating(agonizing or antagonizing) the peptides activity. Antibodies can beprepared against specific fragments containing sites required forfunction or against intact protein that is associated with a cell orcell membrane. See FIG. 2 for structural information relating to theproteins of the present invention.

The invention also encompasses kits for using antibodies to detect thepresence of a protein in a biological sample. The kit can compriseantibodies such as a labeled or labelable antibody and a compound oragent for detecting protein in a biological sample; means fordetermining the amount of protein in the sample; means for comparing theamount of protein in the sample with a standard; and instructions foruse. Such a kit can be supplied to detect a single protein or epitope orcan be configured to detect one of a multitude of epitopes, such as inan antibody detection array. Arrays are described in detail below fornucleic acid arrays and similar methods have been developed for antibodyarrays.

Nucleic Acid Molecules

The present invention further provides isolated nucleic acid moleculesthat encode a kinase peptide or protein of the present invention (cDNA,transcript and genomic sequence). Such nucleic acid molecules willconsist of, consist essentially of, or comprise a nucleotide sequencethat encodes one of the kinase peptides of the present invention, anallelic variant thereof, or an ortholog or paralog thereof.

As used herein, an “isolated” nucleic acid molecule is one that isseparated from other nucleic acid present in the natural source of thenucleic acid. Preferably, an “isolated” nucleic acid is free ofsequences which naturally flank the nucleic acid (i.e., sequenceslocated at the 5′ and 3′ ends of the nucleic acid) in the genomic DNA ofthe organism from which the nucleic acid is derived. However, there canbe some flanking nucleotide sequences, for example up to about 5 KB, 4KB, 3 KB, 2 KB, or 1 KB or less, particularly contiguous peptideencoding sequences and peptide encoding sequences within the same genebut separated by introns in the genomic sequence. The important point isthat the nucleic acid is isolated from remote and unimportant flankingsequences such that it can be subjected to the specific manipulationsdescribed herein such as recombinant expression, preparation of probesand primers, and other uses specific to the nucleic acid sequences.

Moreover, an “isolated” nucleic acid molecule, such as a transcript/cDNAmolecule, can be substantially free of other cellular material, orculture medium when produced by recombinant techniques, or chemicalprecursors or other chemicals when chemically synthesized. However, thenucleic acid molecule can be fused to other coding or regulatorysequences and still be considered isolated.

For example, recombinant DNA molecules contained in a vector areconsidered isolated. Further examples of isolated DNA molecules includerecombinant DNA molecules maintained in heterologous host cells orpurified (partially or substantially) DNA molecules in solution.Isolated RNA molecules include in vivo or in vitro RNA transcripts ofthe isolated DNA molecules of the present invention. Isolated nucleicacid molecules according to the present invention further include suchmolecules produced synthetically.

Accordingly, the present invention provides nucleic acid molecules thatconsist of the nucleotide sequence shown in FIG. 1 or 3 (SEQ ID NO:1,transcript sequence and SEQ ID NO:3, genomic sequence), or any nucleicacid molecule that encodes the protein provided in FIG. 2, SEQ ID NO:2.A nucleic acid molecule consists of a nucleotide sequence when thenucleotide sequence is the complete nucleotide sequence of the nucleicacid molecule.

The present invention turter provides nucleic acid molecules thatconsist essentially of the nucleotide sequence shown in FIG. 1 or 3 (SEQID NO:1, transcript sequence and SEQ ID NO:3, genomic sequence), or anynucleic acid molecule that encodes the protein provided in FIG. 2, SEQID NO:2. A nucleic acid molecule consists essentially of a nucleotidesequence when such a nucleotide sequence is present with only a fewadditional nucleic acid residues in the final nucleic acid molecule.

The present invention further provides nucleic acid molecules thatcomprise the nucleotide sequences shown in FIG. 1 or 3 (SEQ ID NO:1,transcript sequence and SEQ ID NO:3, genomic sequence), or any nucleicacid molecule that encodes the protein provided in FIG. 2, SEQ ID NO:2.A nucleic acid molecule comprises a nucleotide sequence when thenucleotide sequence is at least part of the final nucleotide sequence ofthe nucleic acid molecule. In such a fashion, the nucleic acid moleculecan be only the nucleotide sequence or have additional nucleic acidresidues, such as nucleic acid residues that are naturally associatedwith it or heterologous nucleotide sequences. Such a nucleic acidmolecule can have a few additional nucleotides or can comprises severalhundred or more additional nucleotides. A brief description of howvarious types of these nucleic acid molecules can be readilymade/isolated is provided below.

In FIGS. 1 and 3, both coding and non-coding sequences are provided.Because of the source of the present invention, humans genomic sequence(FIG. 3) and cDNA/transcript sequences (FIG. 1), the nucleic acidmolecules in the Figures will contain genomic intronic sequences, 5′ and3′ non-coding sequences, gene regulatory regions and non-codingintergenic sequences. In general such sequence features are either notedin FIGS. 1 and 3 or can readily be identified using computational toolsknown in the art. As discussed below, some of the non-coding regions,particularly gene regulatory elements such as promoters, are useful fora variety of purposes, e.g. control of heterologous gene expression,target for identifying gene activity modulating compounds, and areparticularly claimed as fragments of the genomic sequence providedherein.

The isolated nucleic acid molecules can encode the mature protein plusadditional amino or carboxyl-terminal amino acids, or amino acidsinterior to the mature peptide (when the mature form has more than onepeptide chain, for instance). Such sequences may play a role inprocessing of a protein from precursor to a mature form, facilitateprotein trafficking, prolong or shorten protein half-life or facilitatemanipulation of a protein for assay or production, among other things.As generally is the case in situ, the additional amino acids may beprocessed away from the mature protein by cellular enzymes.

As mentioned above, the isolated nucleic acid molecules include, but arenot limited to, the sequence encoding the kinase peptide alone, thesequence encoding the mature peptide and additional coding sequences,such as a leader or secretory sequence (e.g., a pre-pro or pro-proteinsequence), the sequence encoding the mature peptide, with or without theadditional coding sequences, plus additional non-coding sequences, forexample introns and non-coding 5′ and 3′ sequences such as transcribedbut non-translated sequences that play a role in transcription, mRNAprocessing (including splicing and polyadenylation signals), ribosomebinding and stability of mRNA. In addition, the nucleic acid moleculemay be fused to a marker sequence encoding, for example, a peptide thatfacilitates purification.

Isolated nucleic acid molecules can be in the form of RNA, such as mRNA,or in the form DNA, including cDNA and genomic DNA obtained by cloningor produced by chemical synthetic techniques or by a combinationthereof. The nucleic acid, especially DNA, can be double-stranded orsingle-stranded. Single-stranded nucleic acid can be the coding strand(sense strand) or the non-coding strand (anti-sense strand).

The invention further provides nucleic acid molecules that encodefragments of the peptides of the present invention as well as nucleicacid molecules that encode obvious variants of the kinase proteins ofthe present invention that are described above. Such nucleic acidmolecules may be naturally occurring, such as allelic variants (samelocus), paralogs (different locus), and orthologs (different organism),or may be constructed by recombinant DNA methods or by chemicalsynthesis. Such non-naturally occurring variants may be made bymutagenesis techniques, including those applied to nucleic acidmolecules, cells, or organisms. Accordingly, as discussed above, thevariants can contain nucleotide substitutions, deletions, inversions andinsertions. Variation can occur in either or both the coding andnon-coding regions. The variations can produce both conservative andnon-conservative amino acid substitutions.

The present invention further provides non-coding fragments of thenucleic acid molecules provided in FIGS. 1 and 3. Preferred non-codingfragments include, but are not limited to, promoter sequences, enhancersequences, gene modulating sequences and gene termination sequences.Such fragments are useful in controlling heterologous gene expressionand in developing screens to identify gene-modulating agents. A promotercan readily be identified as being 5′ to the ATG start site in thegenomic sequence provided in FIG. 3.

A fragment comprises a contiguous nucleotide sequence greater than 12 ormore nucleotides. Further, a fragment could at least 30, 40, 50, 100,250 or 500 nucleotides in length. The length of the fragment will bebased on its intended use. For example, the fragment can encode epitopebearing regions of the peptide, or can be useful as DNA probes andprimers. Such fragments can be isolated using the known nucleotidesequence to synthesize an oligonucleotide probe. A labeled probe canthen be used to screen a cDNA library, genomic DNA library, or mRNA toisolate nucleic acid corresponding to the coding region. Further,primers can be used in PCR reactions to clone specific regions of gene.

A probe/primer typically comprises substantially a purifiedoligonucleotide or oligonucleotide pair. The oligonucleotide typicallycomprises a region of nucleotide sequence that hybridizes understringent conditions to at least about 12, 20, 25, 40, 50 or moreconsecutive nucleotides.

Orthologs, homologs, and allelic variants can be identified usingmethods well known in the art. As described in the Peptide Section,these variants comprise a nucleotide sequence encoding a peptide that istypically 60-70%, 70-80%, 80-90%, and more typically at least about90-95% more homologous to the nucleotide sequence shown in the Figuresheets or a fragment of this sequence. Such nucleic acid molecules canreadily be identified as being able to hybridize under moderate tostringent conditions, to the nucleotide sequence shown in the Figuresheets or a fragment of the sequence. Allelic variants can readily bedetermined by genetic locus of the encoding gene. As indicated in FIG.3, the map position of the kinase gene of the present invention wasdetermined to be on human chromosome 12.

FIG. 3 provides information on SNPs that have been identified at 13different nucleotide positions in the gene encoding the kinase proteinsof the present invention.

As used herein, the term “hybridizes under stringent conditions” isintended to describe conditions for hybridization and washing underwhich nucleotide sequences encoding a peptide at least 60-70% homologousto each other typically remain hybridized to each other. The conditionscan be such that sequences at least about 60%, at least about 70%, or atleast about 80% or more homologous to each other typically remainhybridized to each other. Such stringent conditions are known to thoseskilled in the art and can be found in Current Protocols in MolecularBiology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6. One example ofstringent hybridization conditions are hybridization in 6×sodiumchloride/sodium citrate (SSC) at about 45 C., followed by one or morewashes in 0.2×SSC, 0.1% SDS at 50-65 C. Examples of moderate to lowstringency hybridization conditions are well known in the art.

Nucleic Acid Molecule Uses

The nucleic acid molecules of the present invention are useful forprobes, primers, chemical intermediates, and in biological assays. Thenucleic acid molecules are useful as a hybridization probe for messengerRNA, transcript/cDNA and genomic DNA to isolate full-length cDNA andgenomic clones encoding the peptide described in FIG. 2 and to isolatecDNA and genomic clones that correspond to variants (alleles, orthologs,etc.) producing the same or related peptides shown in FIG. 2. Asillustrated in FIG. 3, SNPs were identified at 13 different nucleotidepositions.

The probe can correspond to any sequence along the entire length of thenucleic acid molecules provided in the Figures. Accordingly, it could bederived from 5′ noncoding regions, the coding region, and 3′ noncodingregions. However, as discussed, fragments are not to be construed asencompassing fragments disclosed prior to the present invention.

The nucleic acid molecules are also useful as primers for PCR to amplifyany given region of a nucleic acid molecule and are useful to synthesizeantisense molecules of desired length and sequence.

The nucleic acid molecules are also useful for constructing recombinantvectors. Such vectors include expression vectors that express a portionof, or all of, the peptide sequences. Vectors also include insertionvectors, used to integrate into another nucleic acid molecule sequence,such as into the cellular genome, to alter in situ expression of a geneand/or gene product. For example, an endogenous coding sequence can bereplaced via homologous recombination with all or part of the codingregion containing one or more specifically introduced mutations.

The nucleic acid molecules are also useful for expressing antigenicportions of the proteins.

The nucleic acid molecules are also useful as probes for determining thechromosomal positions of the nucleic acid molecules by means of in situhybridization methods. As indicated in FIG. 3, the map position of thekinase gene of the present invention was determined to be on humanchromosome 12.

The nucleic acid molecules are also useful in making vectors containingthe gene regulatory regions of the nucleic acid molecules of the presentinvention.

The nucleic acid molecules are also useful for designing ribozymescorresponding to all, or a part, of the mRNA produced from the nucleicacid molecules described herein.

The nucleic acid molecules are also useful for making vectors thatexpress part, or all, of the peptides.

The nucleic acid molecules are also useful for constructing host cellsexpressing a part, or all, of the nucleic acid molecules and peptides.

The nucleic acid molecules are also useful for constructing transgenicanimals expressing all, or a part, of the nucleic acid molecules andpeptides.

The nucleic acid molecules are also useful as hybridization probes fordetermining the presence, level, form and distribution of nucleic acidexpression. Experimental data as provided in FIG. 1 indicates thatkinase proteins of the present invention are expressed in proliferatinghuman erythroid cells of the blood and in glioblastomas of the brain, asindicated by virtual northern blot analysis. Additionally, the tissuesource of the cDNA clone of the present invention indicates expressionin the liver. Accordingly, the probes can be used to detect the presenceof, or to determine levels of, a specific nucleic acid molecule incells, tissues, and in organisms. The nucleic acid whose level isdetermined can be DNA or RNA. Accordingly, probes corresponding to thepeptides described herein can be used to assess expression and/or genecopy number in a given cell, tissue, or organism. These uses arerelevant for diagnosis of disorders involving an increase or decrease inkinase protein expression relative to normal results.

In vitro techniques for detection of mRNA include Northernhybridizations and in situ hybridizations. In vitro techniques fordetecting DNA includes Southern hybridizations and in situhybridization.

Probes can be used as a part of a diagnostic test kit for identifyingcells or tissues that express a kinase protein, such as by measuring alevel of a kinase-encoding nucleic acid in a sample of cells from asubject e.g., mRNA or genomic DNA, or determining if a kinase gene hasbeen mutated. Experimental data as provided in FIG. 1 indicates thatkinase proteins of the present invention are expressed in proliferatinghuman erythroid cells of the blood and in glioblastomas of the brain, asindicated by virtual northern blot analysis. Additionally, the tissuesource of the cDNA clone of the present invention indicates expressionin the liver.

Nucleic acid expression assays are useful for drug screening to identifycompounds that modulate kinase nucleic acid expression.

The invention thus provides a method for identifying a compound that canbe used to treat a disorder associated with nucleic acid expression ofthe kinase gene, particularly biological and pathological processes thatare mediated by the kinase in cells and tissues that express it.Experimental data as provided in FIG. 1 indicates expression in liver,proliferating human erythroid cells of the blood, and glioblastomas ofthe brain. The method typically includes assaying the ability of thecompound to modulate the expression of the kinase nucleic acid and thusidentifying a compound that can be used to treat a disordercharacterized by undesired kinase nucleic acid expression. The assayscan be performed in cell-based and cell-free systems. Cell-based assaysinclude cells naturally expressing the kinase nucleic acid orrecombinant cells genetically engineered to express specific nucleicacid sequences.

The assay for kinase nucleic acid expression can involve direct assay ofnucleic acid levels, such as mRNA levels, or on collateral compoundsinvolved in the signal pathway. Further, the expression of genes thatare up- or down-regulated in response to the kinase protein signalpathway can also be assayed. In this embodiment the regulatory regionsof these genes can be operably linked to a reporter gene such asluciferase.

Thus, modulators of kinase gene expression can be identified in a methodwherein a cell is contacted with a candidate compound and the expressionof mRNA determined. The level of expression of kinase mRNA in thepresence of the candidate compound is compared to the level ofexpression of kinase mRNA in the absence of the candidate compound. Thecandidate compound can then be identified as a modulator of nucleic acidexpression based on this comparison and be used, for example to treat adisorder characterized by aberrant nucleic acid expression. Whenexpression of mRNA is statistically significantly greater in thepresence of the candidate compound than in its absence, the candidatecompound is identified as a stimulator of nucleic acid expression. Whennucleic acid expression is statistically significantly less in thepresence of the candidate compound than in its absence, the candidatecompound is identified as an inhibitor of nucleic acid expression.

The invention further provides methods of treatment, with the nucleicacid as a target, using a compound identified through drug screening asa gene modulator to modulate kinase nucleic acid expression in cells andtissues that express the kinase. Experimental data as provided in FIG. 1indicates that kinase proteins of the present invention are expressed inproliferating human erythroid cells of the blood and in glioblastomas ofthe brain, as indicated by virtual northern blot analysis. Additionally,the tissue source of the cDNA clone of the present invention indicatesexpression in the liver. Modulation includes both up-regulation (i.e.activation or agonization) or down-regulation (suppression orantagonization) or nucleic acid expression.

Alternatively, a modulator for kinase nucleic acid expression can be asmall molecule or drug identified using the screening assays describedherein as long as the drug or small molecule inhibits the kinase nucleicacid expression in the cells and tissues that express the protein.Experimental data as provided in FIG. 1 indicates expression in liver,proliferating human erythroid cells of the blood, and glioblastomas ofthe brain.

The nucleic acid molecules are also useful for monitoring theeffectiveness of modulating compounds on the expression or activity ofthe kinase gene in clinical trials or in a treatment regimen. Thus, thegene expression pattern can serve as a barometer for the continuingeffectiveness of treatment with the compound, particularly withcompounds to which a patient can develop resistance. The gene expressionpattern can also serve as a marker indicative of a physiologicalresponse of the affected cells to the compound. Accordingly, suchmonitoring would allow either increased administration of the compoundor the administration of alternative compounds to which the patient hasnot become resistant. Similarly, if the level of nucleic acid expressionfalls below a desirable level, administration of the compound could becommensurately decreased.

The nucleic acid molecules are also useful in diagnostic assays forqualitative changes in kinase nucleic acid expression, and particularlyin qualitative changes that lead to pathology. The nucleic acidmolecules can be used to detect mutations in kinase genes and geneexpression products such as mRNA. The nucleic acid molecules can be usedas hybridization probes to detect naturally occurring genetic mutationsin the kinase gene and thereby to determine whether a subject with themutation is at risk for a disorder caused by the mutation. Mutationsinclude deletion, addition, or substitution of one or more nucleotidesin the gene, chromosomal rearrangement, such as inversion ortransposition, modification of genomic DNA, such as aberrant methylationpatterns or changes in gene copy number, such as amplification.Detection of a mutated form of the kinase gene associated with adysfunction provides a diagnostic tool for an active disease orsusceptibility to disease when the disease results from overexpression,underexpression, or altered expression of a kinase protein.

Individuals carrying mutations in the kinase gene can be detected at thenucleic acid level by a variety of techniques. FIG. 3 providesinformation on SNPs that have been identified at 13 different nucleotidepositions in the gene encoding the kinase proteins of the presentinvention. As indicated in FIG. 3, the map position of the kinase geneof the present invention was determined to be on human chromosome 12.Genomic DNA can be analyzed directly or can be amplified by using PCRprior to analysis. RNA or cDNA can be used in the same way. In someuses, detection of the mutation involves the use of a probe/primer in apolymerase chain reaction (PCR) (see, e.g. U.S. Pat. Nos. 4,683,195 and4,683,202), such as anchor PCR or RACE PCR, or, alternatively, in aligation chain reaction (LCR) (see, e.g., Landegran et al., Science241:1077-1080 (1988); and Nakazawa et al., PNAS 91:360-364 (1994)), thelatter of which can be particularly useful for detecting point mutationsin the gene (see Abravaya et al., Nucleic Acids Res. 23:675-682 (1995)).This method can include the steps of collecting a sample of cells from apatient, isolating nucleic acid (e.g., genomic, mRNA or both) from thecells of the sample, contacting the nucleic acid sample with one or moreprimers which specifically hybridize to a gene under conditions suchthat hybridization and amplification of the gene (if present) occurs,and detecting the presence or absence of an amplification product, ordetecting the size of the amplification product and comparing the lengthto a control sample. Deletions and insertions can be detected by achange in size of the amplified product compared to the normal genotype.Point mutations can be identified by hybridizing amplified DNA to normalRNA or antisense DNA sequences.

Alternatively, mutations in a kinase gene can be directly identified,for example, by alterations in restriction enzyme digestion patternsdetermined by gel electrophoresis.

Further, sequence-specific ribozymes (U.S. Pat. No. 5,498,531) can beused to score for the presence of specific mutations by development orloss of a ribozyme cleavage site. Perfectly matched sequences can bedistinguished from mismatched sequences by nuclease cleavage digestionassays or by differences in melting temperature.

Sequence changes at specific locations can also be assessed by nucleaseprotection assays such as RNase and S1 protection or the chemicalcleavage method. Furthermore, sequence differences between a mutantkinase gene and a wild-type gene can be determined by direct DNAsequencing. A variety of automated sequencing procedures can be utilizedwhen performing the diagnostic assays (Naeve, C. W., (1995)Biotechniques 19:448), including sequencing by mass spectrometry (see,e.g., PCT International Publication No. WO 94/16101; Cohen et al., Adv.Chromatogr. 36:127-162 (1996); and Griffin et al., Appl. Biochem.Biotechnol. 38:147-159 (1993)).

Other methods for detecting mutations in the gene include methods inwhich protection from cleavage agents is used to detect mismatched basesin RNA/RNA or RNA/DNA duplexes (Myers et al., Science 230:1242 (1985));Cotton et al., PNAS 85:4397 (1988); Saleeba et al., Meth. Enzymol.217:286-295 (1992)), electrophoretic mobility of mutant and wild typenucleic acid is compared (Orita et al., PNAS 86:2766 (1989); Cotton etal., Mutat. Res. 285:125-144 (1993); and Hayashi et al., Genet. Anal.Tech. Appl. 9:73-79 (1992)), and movement of mutant or wild-typefragments in polyacrylamide gels containing a gradient of denaturant isassayed using denaturing gradient gel electrophoresis (Myers et al.,Nature 313:495 (1985)). Examples of other techniques for detecting pointmutations include selective oligonucleotide hybridization, selectiveamplification, and selective primer extension.

The nucleic acid molecules are also useful for testing an individual fora genotype that while not necessarily causing the disease, neverthelessaffects the treatment modality. Thus, the nucleic acid molecules can beused to study the relationship between an individual's genotype and theindividual's response to a compound used for treatment (pharmacogenomicrelationship). Accordingly, the nucleic acid molecules described hereincan be used to assess the mutation content of the kinase gene in anindividual in order to select an appropriate compound or dosage regimenfor treatment. FIG. 3 provides information on SNPs that have beenidentified at 13 different nucleotide positions in the gene encoding thekinase proteins of the present invention.

Thus nucleic acid molecules displaying genetic variations that affecttreatment provide a diagnostic target that can be used to tailortreatment in an individual. Accordingly, the production of recombinantcells and animals containing these polymorphisms allow effectiveclinical design of treatment compounds and dosage regimens.

The nucleic acid molecules are thus useful as antisense constructs tocontrol kinase gene expression in cells, tissues, and organisms. A DNAantisense nucleic acid molecule is designed to be complementary to aregion of the gene involved in transcription, preventing transcriptionand hence production of kinase protein. An antisense RNA or DNA nucleicacid molecule would hybridize to the mRNA and thus block translation ofmRNA into kinase protein.

Alternatively, a class of antisense molecules can be used to inactivatemRNA in order to decrease expression of kinase nucleic acid.Accordingly, these molecules can treat a disorder characterized byabnormal or undesired kinase nucleic acid expression. This techniqueinvolves cleavage by means of ribozymes containing nucleotide sequencescomplementary to one or more regions in the mRNA that attenuate theability of the mRNA to be translated. Possible regions include codingregions and particularly coding regions corresponding to the catalyticand other functional activities of the kinase protein, such as substratebinding.

The nucleic acid molecules also provide vectors for gene therapy inpatients containing cells that are aberrant in kinase gene expression.Thus, recombinant cells, which include the patient's cells that havebeen engineered ex vivo and returned to the patient, are introduced intoan individual where the cells produce the desired kinase protein totreat the individual.

The invention also encompasses kits for detecting the presence of akinase nucleic acid in a biological sample. Experimental data asprovided in FIG. 1 indicates that kinase proteins of the presentinvention are expressed in proliferating human erythroid cells of theblood and in glioblastomas of the brain, as indicated by virtualnorthern blot analysis. Additionally, the tissue source of the cDNAclone of the present invention indicates expression in the liver. Forexample, the kit can comprise reagents such as a labeled or labelablenucleic acid or agent capable of detecting kinase nucleic acid in abiological sample; means for determining the amount of kinase nucleicacid in the sample; and means for comparing the amount of kinase nucleicacid in the sample with a standard. The compound or agent can bepackaged in a suitable container. The kit can further compriseinstructions for using the kit to detect kinase protein mRNA or DNA.

Nucleic Acid Arrays

The present invention further provides nucleic acid detection kits, suchas arrays or microarrays of nucleic acid molecules that are based on thesequence information provided in FIGS. 1 and 3 (SEQ ID NOS:1 and 3).

As used herein “Arrays” or “Microarrays” refers to an array of distinctpolynucleotides or oligonucleotides synthesized on a substrate, such aspaper, nylon or other type of membrane, filter, chip, glass slide, orany other suitable solid support. In one embodiment, the microarray isprepared and used according to the methods described in U.S. Pat. No.5,837,832, Chee et al., PCT application W095/11995 (Chee et al.),Lockhart, D. J. et al. (1996; Nat. Biotech. 14: 1675-1680) and Schena,M. et al. (1996; Proc. Natl. Acad. Sci. 93: 10614-10619), all of whichare incorporated herein in their entirety by reference. In otherembodiments, such arrays are produced by the methods described by Brownet al., U.S. Pat. No. 5,807,522.

The microarray or detection kit is preferably composed of a large numberof unique, single-stranded nucleic acid sequences, usually eithersynthetic antisense oligonucleotides or fragments of cDNAs, fixed to asolid support. The oligonucleotides are preferably about 6-60nucleotides in length, more preferably 15-30 nucleotides in length, andmost preferably about 20-25 nucleotides in length. For a certain type ofmicroarray or detection kit, it may be preferable to useoligonucleotides that are only 7-20 nucleotides in length. Themicroarray or detection kit may contain oligonucleotides that cover theknown 5′, or 3′, sequence, sequential oligonucleotides which cover thefull length sequence; or unique oligonucleotides selected fromparticular areas along the length of the sequence. Polynucleotides usedin the microarray or detection kit may be oligonucleotides that arespecific to a gene or genes of interest.

In order to produce oligonucleotides to a known sequence for amicroarray or detection kit, the gene(s) of interest (or an ORFidentified from the contigs of the present invention) is typicallyexamined using a computer algorithm which starts at the 5′ or at the 3′end of the nucleotide sequence. Typical algorithms will then identifyoligomers of defined length that are unique to the gene, have a GCcontent within a range suitable for hybridization, and lack predictedsecondary structure that may interfere with hybridization. In certainsituations it may be appropriate to use pairs of oligonucleotides on amicroarray or detection kit. The “pairs” will be identical, except forone nucleotide that preferably is located in the center of the sequence.The second oligonucleotide in the pair (mismatched by one) serves as acontrol. The number of oligonucleotide pairs may range from two to onemillion. The oligomers are synthesized at designated areas on asubstrate using a light-directed chemical process. The substrate may bepaper, nylon or other type of membrane, filter, chip, glass slide or anyother suitable solid support.

In another aspect, an oligonucleotide may be synthesized on the surfaceof the substrate by using a chemical coupling procedure and an ink jetapplication apparatus, as described in PCT application W095/251116(Baldeschweiler et al.) which is incorporated herein in its entirety byreference. In another aspect, a “gridded” array analogous to a dot (orslot) blot may be used to arrange and link cDNA fragments oroligonucleotides to the surface of a substrate using a vacuum system,thermal, UV, mechanical or chemical bonding procedures. An array, suchas those described above, may be produced by hand or by using availabledevices (slot blot or dot blot apparatus), materials (any suitable solidsupport), and machines (including robotic instruments), and may contain8, 24, 96, 384, 1536, 6144 or more oligonucleotides, or any other numberbetween two and one million which lends itself to the efficient use ofcommercially available instrumentation.

In order to conduct sample analysis using a microarray or detection kit,the RNA or DNA from a biological sample is made into hybridizationprobes. The mRNA is isolated, and cDNA is produced and used as atemplate to make antisense RNA (aRNA). The aRNA is amplified in thepresence of fluorescent nucleotides, and labeled probes are incubatedwith the microarray or detection kit so that the probe sequenceshybridize to complementary oligonucleotides of the microarray ordetection kit. Incubation conditions are adjusted so that hybridizationoccurs with precise complementary matches or with various degrees ofless complementarity. After removal of nonhybridized probes, a scanneris used to determine the levels and patterns of fluorescence. Thescanned images are examined to determine degree of complementarity andthe relative abundance of each oligonucleotide sequence on themicroarray or detection kit. The biological samples may be obtained fromany bodily fluids (such as blood, urine, saliva, phlegm, gastric juices,etc.), cultured cells, biopsies, or other tissue preparations. Adetection system may be used to measure the absence, presence, andamount of hybridization for all of the distinct sequencessimultaneously. This data may be used for large-scale correlationstudies on the sequences, expression patterns, mutations, variants, orpolymorphisms among samples.

Using such arrays, the present invention provides methods to identifythe expression of the kinase proteins/peptides of the present invention.In detail, such methods comprise incubating a test sample with one ormore nucleic acid molecules and assaying for binding of the nucleic acidmolecule with components within the test sample. Such assays willtypically involve arrays comprising many genes, at least one of which isa gene of the present invention and or alleles of the kinase gene of thepresent invention. FIG. 3 provides information on SNPs that have beenidentified at 13 different nucleotide positions in the gene encoding thekinase proteins of the present invention.

Conditions for incubating a nucleic acid molecule with a test samplevary. Incubation conditions depend on the format employed in the assay,the detection methods employed, and the type and nature of the nucleicacid molecule used in the assay. One skilled in the art will recognizethat any one of the commonly available hybridization, amplification orarray assay formats can readily be adapted to employ the novel fragmentsof the Human genome disclosed herein. Examples of such assays can befound in Chard, T, An Introduction to Radioimmunoassay and RelatedTechniques, Elsevier Science Publishers, Amsterdam, The Netherlands(1986); Bullock, G. R. et al., Techniques in Immunocytochemistry,Academic Press, Orlando, Fla. Vol. 1 (1982), Vol. 2 (1983), Vol. 3(1985); Tijssen, P., Practice and Theory of Enzyme Immunoassays:Laboratory Techniques in Biochemistry and Molecular Biology, ElsevierScience Publishers, Amsterdam, The Netherlands (1985).

The test samples of the present invention include cells, protein ormembrane extracts of cells. The test sample used in the above-describedmethod will vary based on the assay format, nature of the detectionmethod and the tissues, cells or extracts used as the sample to beassayed. Methods for preparing nucleic acid extracts or of cells arewell known in the art and can be readily be adapted in order to obtain asample that is compatible with the system utilized.

In another embodiment of the present invention, kits are provided whichcontain the necessary reagents to carry out the assays of the presentinvention.

Specifically, the invention provides a compartmentalized kit to receive,in close confinement, one or more containers which comprises: (a) afirst container comprising one of the nucleic acid molecules that canbind to a fragment of the Human genome disclosed herein; and (b) one ormore other containers comprising one or more of the following: washreagents, reagents capable of detecting presence of a bound nucleicacid.

In detail, a compartmentalized kit includes any kit in which reagentsare contained in separate containers. Such containers include smallglass containers, plastic containers, strips of plastic, glass or paper,or arraying material such as silica. Such containers allows one toefficiently transfer reagents from one compartment to anothercompartment such that the samples and reagents are notcross-contaminated, and the agents or solutions of each container can beadded in a quantitative fashion from one compartment to another. Suchcontainers will include a container which will accept the test sample, acontainer which contains the nucleic acid probe, containers whichcontain wash reagents (such as phosphate buffered saline, Tris-buffers,etc.), and containers which contain the reagents used to detect thebound probe. One skilled in the art will readily recognize that thepreviously unidentified kinase gene of the present invention can beroutinely identified using the sequence information disclosed herein canbe readily incorporated into one of the established kit formats whichare well known in the art, particularly expression arrays.

Vectors/Host Cells

The invention also provides vectors containing the nucleic acidmolecules described herein. The term “vector” refers to a vehicle,preferably a nucleic acid molecule, which can transport the nucleic acidmolecules. When the vector is a nucleic acid molecule, the nucleic acidmolecules are covalently linked to the vector nucleic acid. With thisaspect of the invention, the vector includes a plasmid, single or doublestranded phage, a single or double stranded RNA or DNA viral vector, orartificial chromosome, such as a BAC, PAC, YAC, OR MAC.

A vector can be maintained in the host cell as an extrachromosomalelement where it replicates and produces additional copies of thenucleic acid molecules. Alternatively, the vector may integrate into thehost cell genome and produce additional copies of the nucleic acidmolecules when the host cell replicates.

The invention provides vectors for the maintenance (cloning vectors) orvectors for expression (expression vectors) of the nucleic acidmolecules. The vectors can function in prokaryotic or eukaryotic cellsor in both (shuttle vectors).

Expression vectors contain cis-acting regulatory regions that areoperably linked in the vector to the nucleic acid molecules such thattranscription of the nucleic acid molecules is allowed in a host cell.The nucleic acid molecules can be introduced into the host cell with aseparate nucleic acid molecule capable of affecting transcription. Thus,the second nucleic acid molecule may provide a trans-acting factorinteracting with the cis-regulatory control region to allowtranscription of the nucleic acid molecules from the vector.Alternatively, a trans-acting factor may be supplied by the host cell.Finally, a trans-acting factor can be produced from the vector itself.It is understood, however, that in some embodiments, transcriptionand/or translation of the nucleic acid molecules can occur in acell-free system.

The regulatory sequence to which the nucleic acid molecules describedherein can be operably linked include promoters for directing mRNAtranscription. These include, but are not limited to, the left promoterfrom bacteriophage λ, the lac, TRP, and TAC promoters from E. coli, theearly and late promoters from SV40, the CMV immediate early promoter,the adenovirus early and late promoters, and retrovirus long-terminalrepeats.

In addition to control regions that promote transcription, expressionvectors may also include regions that modulate transcription, such asrepressor binding sites and enhancers. Examples include the SV40enhancer, the cytomegalovirus immediate early enhancer, polyomaenhancer, adenovirus enhancers, and retrovirus LTR enhancers.

In addition to containing sites for transcription initiation andcontrol, expression vectors can also contain sequences necessary fortranscription termination and, in the transcribed region a ribosomebinding site for translation. Other regulatory control elements forexpression include initiation and termination codons as well aspolyadenylation signals. The person of ordinary skill in the art wouldbe aware of the numerous regulatory sequences that are useful inexpression vectors. Such regulatory sequences are described, forexample, in Sambrook et al., Molecular Cloning: A Laboratory Manual.2nd. ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.,(1989).

A variety of expression vectors can be used to express a nucleic acidmolecule. Such vectors include chromosomal, episomal, and virus-derivedvectors, for example vectors derived from bacterial plasmids, frombacteriophage, from yeast episomes, from yeast chromosomal elements,including yeast artificial chromosomes, from viruses such asbaculoviruses, papovaviruses such as SV40, Vaccinia viruses,adenoviruses, poxviruses, pseudorabies viruses, and retroviruses.Vectors may also be derived from combinations of these sources such asthose derived from plasmid and bacteriophage genetic elements, e.g.cosmids and phagemids. Appropriate cloning and expression vectors forprokaryotic and eukaryotic hosts are described in Sambrook et al.,Molecular Cloning: A Laboratory Manual. 2nd ed., Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., (1989).

The regulatory sequence may provide constitutive expression in one ormore host cells (i.e. tissue specific) or may provide for inducibleexpression in one or more cell types such as by temperature, nutrientadditive, or exogenous factor such as a hormone or other ligand. Avariety of vectors providing for constitutive and inducible expressionin prokaryotic and eukaryotic hosts are well known to those of ordinaryskill in the art.

The nucleic acid molecules can be inserted into the vector nucleic acidby well-known methodology. Generally, the DNA sequence that willultimately be expressed is joined to an expression vector by cleavingthe DNA sequence and the expression vector with one or more restrictionenzymes and then ligating the fragments together. Procedures forrestriction enzyme digestion and ligation are well known to those ofordinary skill in the art.

The vector containing the appropriate nucleic acid molecule can beintroduced into an appropriate host cell for propagation or expressionusing well-known techniques. Bacterial cells include, but are notlimited to, E. coli, Streptomyces, and Salmonella typhimurium.Eukaryotic cells include, but are not limited to, yeast, insect cellssuch as Drosophila, animal cells such as COS and CHO cells, and plantcells.

As described herein, it may be desirable to express the peptide as afusion protein. Accordingly, the invention provides fusion vectors thatallow for the production of the peptides. Fusion vectors can increasethe expression of a recombinant protein, increase the solubility of therecombinant protein, and aid in the purification of the protein byacting for example as a ligand for affinity purification. A proteolyticcleavage site may be introduced at the junction of the fusion moiety sothat the desired peptide can ultimately be separated from the fusionmoiety. Proteolytic enzymes include, but are not limited to, factor Xa,thrombin, and enterokinase. Typical fusion expression vectors includepGEX (Smith et al., Gene 67:31-40 (1988)), pMAL (New England Biolabs,Beverly, Mass.) and pRIT5 (Pharmacia, Piscataway, N.J.) which fuseglutathione S-transferase (GST), maltose E binding protein, or proteinA, respectively, to the target recombinant protein. Examples of suitableinducible non-fusion E. coli expression vectors include pTrc (Amann etal., Gene 69:301-315 (1988)) and pET 11 d (Studier et al., GeneExpression Technology: Methods in Enzymology 185:60-89 (1990)).

Recombinant protein expression can be maximized in host bacteria byproviding a genetic background wherein the host cell has an impairedcapacity to proteolytically cleave the recombinant protein. (Gottesman,S., Gene Expression Technology: Methods in Enzymology 185, AcademicPress, San Diego, Calif. (1990)119-128). Alternatively, the sequence ofthe nucleic acid molecule of interest can be altered to providepreferential codon usage for a specific host cell, for example E. coli.(Wada et al., Nucleic Acids Res. 20:2111-2118 (1992)).

The nucleic acid molecules can also be expressed by expression vectorsthat are operative in yeast. Examples of vectors for expression in yeaste.g., S. cerevisiae include pYepSec1 (Baldari, et al., EMBO J. 6:229-234(1987)), pMFa (Kurjan et al., Cell 30:933-943(1982)), pJRY88 (Schultz etal., Gene 54:113-123 (1987)), and pYES2 (Invitrogen Corporation, SanDiego, Calif.).

The nucleic acid molecules can also be expressed in insect cells using,for example, baculovirus expression vectors. Baculovirus vectorsavailable for expression of proteins in cultured insect cells (e.g., Sf9 cells) include the pAc series (Smith et al., Mol. Cell Biol.3:2156-2165 (1983)) and the pVL series (Lucklow et al., Virology170:31-39 (1989)).

In certain embodiments of the invention, the nucleic acid moleculesdescribed herein are expressed in mammalian cells using mammalianexpression vectors. Examples of mammalian expression vectors includepCDM8 (Seed, B. Nature 329:840(1987)) and pMT2PC (Kaufman et al., EMBOJ. 6:187-195 (1987)).

The expression vectors listed herein are provided by way of example onlyof the well-known vectors available to those of ordinary skill in theart that would be useful to express the nucleic acid molecules. Theperson of ordinary skill in the art would be aware of other vectorssuitable for maintenance propagation or expression of the nucleic acidmolecules described herein.

These are found for example in Sambrook, J., Fritsh, E. F., andManiatis, T. Molecular Cloning: A Laboratory Manual. 2nd, ed., ColdSpring Harbor Laboratory, Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y., 1989.

The invention also encompasses vectors in which the nucleic acidsequences described herein are cloned into the vector in reverseorientation, but operably linked to a regulatory sequence that permitstranscription of antisense RNA. Thus, an antisense transcript can beproduced to all, or to a portion, of the nucleic acid molecule sequencesdescribed herein, including both coding and non-coding regions.Expression of this antisense RNA is subject to each of the parametersdescribed above in relation to expression of the sense RNA (regulatorysequences, constitutive or inducible expression, tissue-specificexpression).

The invention also relates to recombinant host cells containing thevectors described herein. Host cells therefore include prokaryoticcells, lower eukaryotic cells such as yeast, other eukaryotic cells suchas insect cells, and higher eukaryotic cells such as mammalian cells.

The recombinant host cells are prepared by introducing the vectorconstructs described herein into the cells by techniques readilyavailable to the person of ordinary skill in the art. These include, butare not limited to, calcium phosphate transfection,DEAE-dextran-mediated transfection, cationic lipid-mediatedtransfection, electroporation, transduction, infection, a lipofection,and other techniques such as those found in Sambrook, et al. (MolecularCloning: A Laboratory Manual. 2nd, ed, Cold Spring Harbor Laboratory,Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989).

Host cells can contain more than one vector. Thus, different nucleotidesequences can be introduced on different vectors of the same cell.Similarly, the nucleic acid molecules can be introduced either alone orwith other nucleic acid molecules that are not related to the nucleicacid molecules such as those providing trans-acting factors forexpression vectors. When more than one vector is introduced into a cell,the vectors can be introduced independently, co-introduced or joined tothe nucleic acid molecule vector.

In the case of bacteriophage and viral vectors, these can be introducedinto cells as packaged or encapsulated virus by standard procedures forinfection and transduction. Viral vectors can be replication-competentor replication-defective. In the case in which viral replication isdefective, replication will occur in host cells providing functions thatcomplement the defects.

Vectors generally include selectable markers that enable the selectionof the subpopulation of cells that contain the recombinant vectorconstructs. The marker can be contained in the same vector that containsthe nucleic acid molecules described herein or may be on a separatevector. Markers include tetracycline or ampicillin-resistance genes forprokaryotic host cells and dihydrofolate reductase or neomycinresistance for eukaryotic host cells. However, any marker that providesselection for a phenotypic trait will be effective.

While the mature proteins can be produced in bacteria, yeast, mammaliancells, and other cells under the control of the appropriate regulatorysequences, cell-free transcription and translation systems can also beused to produce these proteins using RNA derived from the DNA constructsdescribed herein.

Where secretion of the peptide is desired, which is difficult to achievewith multi-transmembrane domain containing proteins such as kinases,appropriate secretion signals are incorporated into the vector. Thesignal sequence can be endogenous to the peptides or heterologous tothese peptides.

Where the peptide is not secreted into the medium, which is typicallythe case with kinases, the protein can be isolated from the host cell bystandard disruption procedures, including freeze thaw, sonication,mechanical disruption, use of lysing agents and the like. The peptidecan then be recovered and purified by well-known purification methodsincluding ammonium sulfate precipitation, acid extraction, anion orcationic exchange chromatography, phosphocellulose chromatography,hydrophobic-interaction chromatography, affinity chromatography,hydroxylapatite chromatography, lectin chromatography, or highperformance liquid chromatography.

It is also understood that depending upon the host cell in recombinantproduction of the peptides described herein, the peptides can havevarious glycosylation patterns, depending upon the cell, or maybenon-glycosylated as when produced in bacteria. In addition, the peptidesmay include an initial modified methionine in some cases as a result ofa host-mediated process.

Uses of Vectors and Host Cells

The recombinant host cells expressing the peptides described herein havea variety of uses. First, the cells are useful for producing a kinaseprotein or peptide that can be further purified to produce desiredamounts of kinase protein or fragments. Thus, host cells containingexpression vectors are useful for peptide production.

Host cells are also useful for conducting cell-based assays involvingthe kinase protein or kinase protein fragments, such as those describedabove as well as other formats known in the art. Thus, a recombinanthost cell expressing a native kinase protein is useful for assayingcompounds that stimulate or inhibit kinase protein function.

Host cells are also useful for identifying kinase protein mutants inwhich these functions are affected. If the mutants naturally occur andgive rise to a pathology, host cells containing the mutations are usefulto assay compounds that have a desired effect on the mutant kinaseprotein (for example, stimulating or inhibiting function) which may notbe indicated by their effect on the native kinase protein.

Genetically engineered host cells can be further used to producenon-human transgenic animals. A transgenic animal is preferably amammal, for example a rodent, such as a rat or mouse, in which one ormore of the cells of the animal include a transgene. A transgene isexogenous DNA which is integrated into the genome of a cell from which atransgenic animal develops and which remains in the genome of the matureanimal in one or more cell types or tissues of the transgenic animal.These animals are useful for studying the function of a kinase proteinand identifying and evaluating modulators of kinase protein activity.Other examples of transgenic animals include non-human primates, sheep,dogs, cows, goats, chickens, and amphibians.

A transgenic animal can be produced by introducing nucleic acid into themale pronuclei of a fertilized oocyte, e.g., by microinjection,retroviral infection, and allowing the oocyte to develop in apseudopregnant female foster animal. Any of the kinase proteinnucleotide sequences can be introduced as a transgene into the genome ofa non-human animal, such as a mouse.

Any of the regulatory or other sequences useful in expression vectorscan form part of the transgenic sequence. This includes intronicsequences and polyadenylation signals, if not already included. Atissue-specific regulatory sequence(s) can be operably linked to thetransgene to direct expression of the kinase protein to particularcells.

Methods for generating transgenic animals via embryo manipulation andmicroinjection, particularly animals such as mice, have becomeconventional in the art and are described, for example, in U.S. Pat.Nos. 4,736,866 and 4,870,009, both by Leder et al., U.S. Pat. No.4,873,191 by Wagner et al. and in Hogan, B., Manipulating the MouseEmbryo, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.,1986). Similar methods are used for production of other transgenicanimals. A transgenic founder animal can be identified based upon thepresence of the transgene in its genome and/or expression of transgenicmRNA in tissues or cells of the animals. A transgenic founder animal canthen be used to breed additional animals carrying the transgene.Moreover, transgenic animals carrying a transgene can further be bred toother transgenic animals carrying other transgenes. A transgenic animalalso includes animals in which the entire animal or tissues in theanimal have been produced using the homologously recombinant host cellsdescribed herein.

In another embodiment, transgenic non-human animals can be producedwhich contain selected systems that allow for regulated expression ofthe transgene. One example of such a system is the credloxP recombinasesystem of bacteriophage P1. For a description of the cre/loxPrecombinase system, see, e.g., Lakso et al. PNAS 89:6232-6236 (1992).Another example of a recombinase system is the FLP recombinase system ofS. cerevisiae (O'Gorman et al. Science 251:1351-1355 (1991). If acre/loxP recombinase system is used to regulate expression of thetransgene, animals containing transgenes encoding both the Crerecombinase and a selected protein is required. Such animals can beprovided through the construction of “double” transgenic animals, e.g.,by mating two transgenic animals, one containing a transgene encoding aselected protein and the other containing a transgene encoding arecombinase.

Clones of the non-human transgenic animals described herein can also beproduced according to the methods described in Wilmut, I. et al. Nature385:810-813 (1997) and PCT International Publication Nos. WO 97/07668and WO 97/07669. In brief, a cell, e.g., a somatic cell, from thetransgenic animal can be isolated and induced to exit the growth cycleand enter G_(o) phase. The quiescent cell can then be fused, e.g.,through the use of electrical pulses, to an enucleated oocyte from ananimal of the same species from which the quiescent cell is isolated.The reconstructed oocyte is then cultured such that it develops tomorula or blastocyst and then transferred to pseudopregnant femalefoster animal. The offspring born of this female foster animal will be aclone of the animal from which the cell, e.g., the somatic cell, isisolated.

Transgenic animals containing recombinant cells that express thepeptides described herein are useful to conduct the assays describedherein in an in vivo context. Accordingly, the various physiologicalfactors that are present in vivo and that could effect substratebinding, kinase protein activation, and signal transduction, may not beevident from in vitro cell-free or cell-based assays. Accordingly, it isuseful to provide non-human transgenic animals to assay in vivo kinaseprotein function, including substrate interaction, the effect ofspecific mutant kinase proteins on kinase protein function and substrateinteraction, and the effect of chimeric kinase proteins. It is alsopossible to assess the effect of null mutations, that is, mutations thatsubstantially or completely eliminate one or more kinase proteinfunctions.

All publications and patents mentioned in the above specification areherein incorporated by reference. Various modifications and variationsof the described method and system of the invention will be apparent tothose skilled in the art without departing from the scope and spirit ofthe invention. Although the invention has been described in connectionwith specific preferred embodiments, it should be understood that theinvention as claimed should not be unduly limited to such specificembodiments. Indeed, various modifications of the above-described modesfor carrying out the invention which are obvious to those skilled in thefield of molecular biology or related fields are intended to be withinthe scope of the following claims.

6 1 1133 DNA Human 1 gcggggcgga acagatcgca gacctggggg ttcgcagagccgccagtggg gagatgttga 60 agttcaaata tggagcgcgg aatcctttgg atgctggtgctgctgaaccc attgccagcc 120 gggcctccag gctgaatctg ttcttccagg ggaaaccaccctttatgact caacagcaga 180 tgtctcctct ttcccgagaa gggatattag atgccctctttgttctcttt gaagaatgca 240 gtcagcctgc tctgatgaag attaagcacg tgagcaactttgtccggaag tattccgaca 300 ccatagctga gttacaggag ctccagcctt cggcaaaggacttcgaagtc agaagtcttg 360 taggttgtgg tcactttgct gaagtgcagg tggtaagagagaaagcaacc ggggacatct 420 atgctatgaa agtgatgaag aagaaggctt tattggcccaggagcaggtt tcattttttg 480 aggaagagcg gaacatatta tctcgaagca caagcccgtggatcccccaa ttacagtatg 540 cctttcagga caaaaatcac ctttatctgg tcatggaatatcagcctgga ggggacttgc 600 tgtcactttt gaatagatat gaggaccagt tagatgaaaacctgatacag ttttacctag 660 ctgagctgat tttggctgtt cacagcgttc atctgatgggatacgtgcat cgagacatca 720 agcctgagaa cattctcgtt gaccgcacag gacacatcaagctggtggat tttggatctg 780 ccgcgaaaat gaattcaaac aagatggtaa aaaatggaataagatagctt aatagagttt 840 atactaaaaa gtgttcttgg tcctcctaag tttgggaagtgttgggataa aatggtgaac 900 aatgttttgg agcctttggc agtgtatggg ggtggggacagggacacaga accatttccc 960 agaccgtggc acctttttat ttatagtgcc tgttaataccctccaagaca tttttaggag 1020 cattgttata gtttggttag aaataaagga aaatgcttaaaaaaaaaaaa aaaaaaaaaa 1080 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaaaaaaaaaaaa aaa 1133 2 257 PRT Human 2 Met Leu Lys Phe Lys Tyr Gly AlaArg Asn Pro Leu Asp Ala Gly Ala 1 5 10 15 Ala Glu Pro Ile Ala Ser ArgAla Ser Arg Leu Asn Leu Phe Phe Gln 20 25 30 Gly Lys Pro Pro Phe Met ThrGln Gln Gln Met Ser Pro Leu Ser Arg 35 40 45 Glu Gly Ile Leu Asp Ala LeuPhe Val Leu Phe Glu Glu Cys Ser Gln 50 55 60 Pro Ala Leu Met Lys Ile LysHis Val Ser Asn Phe Val Arg Lys Tyr 65 70 75 80 Ser Asp Thr Ile Ala GluLeu Gln Glu Leu Gln Pro Ser Ala Lys Asp 85 90 95 Phe Glu Val Arg Ser LeuVal Gly Cys Gly His Phe Ala Glu Val Gln 100 105 110 Val Val Arg Glu LysAla Thr Gly Asp Ile Tyr Ala Met Lys Val Met 115 120 125 Lys Lys Lys AlaLeu Leu Ala Gln Glu Gln Val Ser Phe Phe Glu Glu 130 135 140 Glu Arg AsnIle Leu Ser Arg Ser Thr Ser Pro Trp Ile Pro Gln Leu 145 150 155 160 GlnTyr Ala Phe Gln Asp Lys Asn His Leu Tyr Leu Val Met Glu Tyr 165 170 175Gln Pro Gly Gly Asp Leu Leu Ser Leu Leu Asn Arg Tyr Glu Asp Gln 180 185190 Leu Asp Glu Asn Leu Ile Gln Phe Tyr Leu Ala Glu Leu Ile Leu Ala 195200 205 Val His Ser Val His Leu Met Gly Tyr Val His Arg Asp Ile Lys Pro210 215 220 Glu Asn Ile Leu Val Asp Arg Thr Gly His Ile Lys Leu Val AspPhe 225 230 235 240 Gly Ser Ala Ala Lys Met Asn Ser Asn Lys Met Val LysAsn Gly Ile 245 250 255 Arg 3 48763 DNA Human 3 gggtgacgga gtgagattctgtctaagaaa aaagaaaaaa aaagaggtgc ttgataaata 60 gtagctatcc attattggccccgggaacaa gaagtaagtt atgtttgggg aaggaaaaaa 120 gaacaaatgt gtattaagcaagcctgtagc tctaattatg tgctggtgtg cgtgtgtgtg 180 tgtgtgtgtg tgagagagagaacacatctc cagttctgtc tactgtagaa ttaggagagt 240 acaaaaagga ctttacatatataaatagaa catacacaca cacacatgcg tgcacacata 300 tacacacaat ttaatcattatgaaaccaca tccatattgt tgctacctag gttaagaaat 360 agatcacagc agcaccccaacaccctgaaa ggcctccatc ccaaccccag gtaactacta 420 ttctggctgt tgctttctttatggttttgt cattacttta aacaatgaca aaaactgcaa 480 tgatttgcat caacctaatacatccctcct taaacaatgt tgctttgttt tgtcctgttt 540 tggaacttat aagaatggaatcataatgga atcatatgtt attttcttgc ttccttcatt 600 aggccttgtt ttgagactcattatgtcatt gtggttagtt gcagtttatt ctttttcatt 660 gcttgtgaaa acactgcaatatacaatttt gtcttttcta ctgctgatgg acatttatat 720 cacttccagt tttttgcgaacactattttg tattcttata cacatctctt ggtgtacata 780 agtaggagtt tctcgccggcgtggtggctc agggcctgta atctcagcac tttgggaggc 840 cgaggtgggc agatcactcgaggtcaggag ttcaagacca gcctggccaa cacggtgaaa 900 ccccatctct actaaaaatacaaacaattg ggcatggtgg catgcacctg taatcccagt 960 tacttgggag gatgagacaagagaatagct tgaacctggg aggtggaggt tgcagtgagg 1020 cgagatcgtg ccattgcactccagcctggg agacagagca agactccatc tcaaaataaa 1080 taaataaata ggagtttttcttaggtagag aaactacacc tagcaatagt catagaatgc 1140 acaaatcttc aatgttagcaaataatgcca aacttttttt tcaaatttca aagagattgt 1200 atccatttac acgcctacgggtactgtata agtgtgtgta cttccacatc ttcgcaaaca 1260 ctgtcacatc cttttgttgttgttgttctc gaatttgagt gttattcttt ctcactgtga 1320 ctttattttt catattttctgattatgaac gaggttgaca actttcacac atttgttggt 1380 catctggatt tcctttttggtgaagtgcct gtttaagtat ctcgtctata atttatttta 1440 aagtgtcctt tcagacagtctcaatgactg tcaccaactc cttgcagggc agtcagcccg 1500 gagatagagt aatcaaggtaggttgaagtc aagctcaaaa cattcgctgc ctcagctgta 1560 gcagaggacc actgggcttccccaggtaac aagtacttct accttagcca catgagagag 1620 aaagaagacc aggcagagcagcctggctgc cttcctcctt gcaggtggcc gagagcaggg 1680 gacagcgccc tggcgacctcctcagggatc ctagattaac agtcgcgtcc tcaaacgcag 1740 catcctgcgt aaccgccaatttcaaacttc caagacctgc cctgctgatt ttgcccttcc 1800 ctttttcccg ttggtcgcgagtcaaaggaa gatgcaattt gattggctct ccccttcact 1860 ttcctccatg cctttagggacatgggcggg gcctggctga gacgcccatg tctatcatag 1920 gagcggagac gctgattggtccaaacacgg ctgagacccg cccgcgccgt tcctcgggtt 1980 caaacgcggc ggcgggaggcgcggggcgga acagatcgca gacctggggg ttcgcagagc 2040 gtgagtctga tcccccagacccaattctac cgcacccggc tctgcaaggc caggggaggg 2100 ccgcctccac ccatacaagtcccgggtttc cctcccgccc cggggagggc ggcgattcca 2160 cccccagggc tgcgggaggcctggagggtc ttccggggct agctgtgcgc gcgcccacct 2220 tccttgggag ccgaggggtcagccgagtgg tgctggggca ggaggcttgc tcctccccta 2280 aaccaggcgg agtgctttgtctcttcagct ctgcctcctg tcagcactaa ctgcattatt 2340 ctgcccagtg tagtcggccggttccttatt atctgcgtga acttagccat ttacttaacc 2400 tctctgtttc agcgtattcataccccgtgc ccaccccatc acctcatgat gcccccgcct 2460 ctttcgctct gctccagtccgtctggcctc gctgttgctg gagaggccag gtcctgcctc 2520 agtgcttttg gcttggctgtttcgtttgcc acggatgtct ttctttcccc agatatcaac 2580 atggcttgct ggtcattcgcttcaggtctt caagtcttgg gtcaaatggt ggcttctcag 2640 tgaagtctta tttgaccacactaaaaattg caccatctca cccccattgt ccttttcttg 2700 ctcgattttg tttttaccccatagcactta acaccttaca acaagctata tattttgctt 2760 atttcagtca ttcatttaataactattcgc acctatttgt gtgccaggct atgtgtgccc 2820 ccactgcatg ggggcaaacatctctgccct tgtggagctt ccattctaag gggggagata 2880 ataaacacat ttataagtaagagagtatgt cagataagtg tatcatctcc tgtcacagtg 2940 agttaaaatc tggtgtttaatctccatgat tagactgagc ttcctaaaac tggagtggta 3000 gctgattttc acctccttgtccctgatatc ttgagggaga tcaggatctc tcaggccctt 3060 cctgctcaaa acataggacacacttgactt ttctgatatc ctttcagcgc cagtggggag 3120 atgttgaagt tcaaatatggagcgcggaat cctttggatg ctggtgctgc tgaacccatt 3180 gccagccggg cctccaggctgaatctgttc ttccaggtaa cagcctaccc tgccaacttt 3240 gctcacctgt gtgtgtccttggaatctcct tgtcactcac ctttgctttt atttatttgt 3300 ttatttattt agagtctcagtctctcaggc tggagtacag tggtgcaatc tcagctcact 3360 gcaacctccg cctcctgggttcaagcgatt ctcctgcctc agcctccaga gtagctggga 3420 ctacagccgc ctgccaccacacccggctaa attttgtatt tttcttttta gtagagacgg 3480 ggtttcacca tgttggccaggctagggtcg aactcctgac ctcaagtgat ccacctgcct 3540 tggcctccta aagtgctgggattacaggca tgaaccgtgc ccagcttgct tttattatag 3600 gaccagggat aatattttaggggaaattct gttttgtttt gtttgaaaca aggtcttctg 3660 tcgactctag gcctgtgccaccatgcctgg ctaatttttt aattttttgt agggatgggg 3720 tctcactgtg ttgcccaggctgatatagaa cacctgactt caagtgagcc tcttgccttg 3780 gcctcccaaa gcactggggttataggtgtg agccactgca cctggccctc tatttagagt 3840 tttatatgca ctgattcttttggaaaaaag acactgtgca gaagtagata gctgaacttg 3900 ccttagaagg gagatcttttcatatttctc acactttaca cttctgtact aaagtttatt 3960 cattcattga ttgattggttgcttgcaaga cagggtcttg ctctgtggct caggctggag 4020 tgcattggca caatcacggcttactgcagc cttgacctcc tgggctcaaa cgatcctccc 4080 acttcagctt cctgagtagctgggaccaca ggtgtgtgcc accatacctg gctaattttt 4140 gtattttttg tagagatgaggtttcaccat gttgcccagg caggtctcga attcctgggc 4200 tcaagtgatc tacttgtcacagcttctgca agtgttgggc ttacaggcat aagcccctgt 4260 accagggcaa gtttgtccttttattgaaga aagaaaaata aatgaacaaa gatgcttttt 4320 aaaactacaa tttctgtgggtataatccta ttcattttca ttgcagggat gtttattttt 4380 taagattttt tttttttttttttgagacag agtcttcgct gtcgcccagg ctggagtgca 4440 gtggcgcgat ctcggctcactgcaggctct gccccccggg gttcacgcca ttctcctgcc 4500 tcagcctccc acgtagctgggactacaggc gcccgtcacc tcgcccggct aattttttgt 4560 atttttagta gagacggggtttcactgtgt tagccaggat ggtatttttt aagattttaa 4620 aaaaagtttt gatgaataccacacctgttt aaccctcatt cctctcaaga tacacatttc 4680 tgtcacccca gatgcgttaaaacttaatat cataagatta cttccaaata gatttttaat 4740 tcttttgttt ctgatgtatgtggaacactg gtgaagtaga aatccttgtt tgatttatgt 4800 attcgtaagt cagggggacaatagagacca tgaagattta gaattgaatc ccagtcccag 4860 cactagttag ctgcattactttgggtgagt cagttacctt ttctgagtcc atttgctatt 4920 ctttaaaata ggttgtagcctgtaatgcca gtattttcgg aggctgaggc gggcggatta 4980 cttgaggtca cgggttcgagaccagcctgg acaacgtggt gaaaccctgt ctctactaaa 5040 aatatagaaa attagctgggcatggtggtc gcatgtacct gtaatcccag ctacttgaaa 5100 agctgaagca ggagaatcatttgaacccgg gaggcggagg ttgtcgtgag ccgagatggt 5160 gcactgcact ccagcctgggcgacagagtg ggtaagactc catctcaaaa caaaacaaaa 5220 caaaagaaaa caaaaaaaataacatagagg ttgtagtacc taatccacag ggttgttgtg 5280 aggattagat gagatattcgatttaaagca cttagcacct tgcctggctc ttagtaaact 5340 ccttataaaa aatggtaattattgttaata ctcagcatag aatagtatta gttataatat 5400 taatactaaa tttgtttccttaatagtaat tatatttggg aaggtagtta tgtaggatac 5460 ctgtaagatg atgaatgatgaagtattctt gataactttt tttttttttc caaaatattg 5520 gtattgggtg tttaaacagatgagagtgga aacaaattga aagcttaggt ttttctgtgg 5580 gaccatcccc atcagcattttaagtcttga catatctttc acaaatgaat agtctgtctt 5640 taaccttaga tggctggagtgctgccacgt ttcagcccct ttatcatgct actttaaaat 5700 atctccaact tgctgggcgtggtggctcac gcctgtaatc ctagcaattt gggaggctga 5760 ggtgggtgga ttgcttgaggtcaggagttc gagagcagcc cgggcaacat ggtgagcccc 5820 tccgtttcta ctaaaaacacaaaaaatagc tgactgtgat ggtgtgtgcc tgtagtccca 5880 gctactcggg aggctgaggcaggaggatca cttgagccct agaggcagag gttgcagtga 5940 gctaagattg tgccactgcacttcagcact tcagcctagg cgacagagca agaccctgta 6000 aattaaaaaa aaaaaaaaaaagaaaaggaa aaaaatttcc aacttattaa gggcttatag 6060 tgtgctgatt atgtaatagttatggcttcc aatgtgtctg gcatagaact ggcatgtttc 6120 tgagtatctc acttcagcctcatgacagag gtaaggacta tttttaattt aaactttaaa 6180 taggaggcaa caggccaggtgtggtggctc acacctgtaa tcccagtact ttgggaggct 6240 gaggcaggtg gattgcttgagtccaagagt tcaagactag cctgggcaaa atggtgaaac 6300 cccatctcta caaaaaatataaataattag tcaggcatgg cggtgtgtgc ctgtagtccc 6360 agctactcag gaggctgaggtgggggcatc tctggggccc cggaggcaga ggttgtagtg 6420 agttgagatt gcaacactgcactccagcct gggcaacaga acgagaccct gtttctaaat 6480 aaatacataa ataggaggcaacagatatag acagatatgg aggtaggtaa ggccttgccc 6540 aagatcatac acgttgggttttgcagatga ggccaagatc agactccatc tttggttggt 6600 ctgactccaa aggctgaccacatagccatt gggccacagc acctgtgcac gtcagaattt 6660 attaagtata tcttgtatttagtcattata acaggaagac ttatgggtaa accctcagtt 6720 catctctttt taatgctgagatccccctgc ccagtaaagc tattattgca agtatagtat 6780 atacctatca tttgccttgagttatcaggt aaggatgctg tttgttcttt tcccatatag 6840 tgctgtttga atgaggttgagatacagtag caattttgtt ttccattcag gtgagtacct 6900 tagactgagt gtcattttgtcttttttact tctactcaac aggatttcct gacatgttcg 6960 aggtcagtga ttgtcagactttctgagcca gcaaaatttc ccaaattgct gggtagacac 7020 aggttttcca actttttattttgccaagta aggatatata aaaaaaaaat aaaaagaaag 7080 acctattatt ttctggcccttgtatttcat aaagggcatt ttaagaaaca acaagacagg 7140 aagaacatca tctcagaataaaggaccatt tttaaatttg aatacattta gttttataaa 7200 aaagatatca tgtggtgttcattttttctc atttcactgc aggctgttga aaactttgtt 7260 aagaaccagt actatatttgggaacccctg ctttaattga tctaaactct tgaagaatag 7320 aagaaacaaa gcattttatttttctgagtt actggcaact attactaaag tgacagatat 7380 ggtggccttg aatgcagtgcttcccaaacc tgattgaggt ctgactctct tggggaccag 7440 ggtctcattc tgttgcccaggctggagtgt ggcagcacaa tcttggctca ctgcagcctt 7500 tacttcttgg gctcaagtgatccttctacc tcagtctcac aagtggctag gactacagga 7560 ccatggcact acacctggctaatttttttt tgtttgtttg tagagatggg atctcgctgt 7620 gttgccctgg ctggtcttgaactcctgggc tcaagtgatc ctcccacctt ggcctcccaa 7680 agtgctagta ttccaggtgtgagccacctc tccctgctgg ggaacttgtt aataaaacag 7740 attctaggct acagtctggaaaattctaat tcatttggtt gtgggggagg ggggcatagg 7800 accagagaat gtgtttgtttgtttgtttgt ttttcttaaa ttctccagtg ctgttgtgat 7860 tcaaatgcag ccggtctgtttctgttatca agtgctgtgt aacaaagcac tcacaaagtt 7920 taaagcaaca atgatttattttttcttagg attctgtggg ttggctggac tcagctaggt 7980 agttctgctt catcctgtgatgtcagctgg ggtcacttgt ggggctacat tcagctggga 8040 ttatgtctgg gactggaacatgtgggtgct gactgctggc tggggcacct tagtgtttct 8100 cacatggcct ctcttctccatgaggtcttt cagtagtata gcccaggact cgtaactttt 8160 tttttttttt taagacagactgtcgccctg tcgcccaggc tggagtgcag tggcacgatc 8220 tctgctcact gcaacctccgcctcctgggt tcaagcaatt ctcctgcccc agcctcccga 8280 gtagctggga ttacaggcacgtgcctccac gcccggctaa tgtttgcatt tttagtagag 8340 atggggtttc accacgttggtcaggctggt ctcgaacttc tgacctcgcg atccgcctgc 8400 ctcggcctcc caaagtgttggaattacagg tgtgagccac tgcacctggc cgactcgtaa 8460 ctttttttgt aagtaataaatattttaggc tttgtgggtc ctgtagtctc tgttgcaacc 8520 actcaacttg gccatggtagcacaaaagca gctaaagaca atatgtaaat gatgggtgta 8580 gctgtgttcc agtaaaacttataaaaagtc cgtgggctgg atttggtcca agggctacag 8640 attgcacacc cctggtctagcccaagcatc tgtgcatggt ggctggcttc ccaaaagtgg 8700 aagctgctaa gctgcctttttttttttttt tttttttttt gagagggagt ctcactgtgt 8760 tgcctaggct ggagtgcggtggtgtgatct cggctcactg caacctccat ctcccgggtg 8820 caggcaattc tcatgcctcaacctcccagg tagctgggat tacgggtgcc taccaccacg 8880 cctggctaat ttttgtattttggtagagac agggtttcac catgttggcc aggctggtct 8940 caaactcctg acctcaagtgatccacccgt cttggcctcc caaagtgctg ggattacaga 9000 tgtgagccac cgtgtctggccgcttgacaa gcttcttaaa ggcactgccc tgaactggca 9060 cagtgtcact tgtgtcacattcttttggtt gaagagagtc tcagagatgg cacagattca 9120 aaggcaggag aaatagactccagcgcttaa agtaaggagt agcatgtgcc tacagaattg 9180 gaggaactgt tggaggccatctttgaagag agaccaccac tatccatggc ttggcacgtg 9240 ggaatcactg ctctataccagggttgcaga ctcatgtctt tgggggccag gcagtgagta 9300 taaatgagtc aagtgggccagttggaagat ggagtcagac ctgcagtgaa ctcccaaaca 9360 catctgctac cgggaggggcagcattactc agctccagct cagcgtcatc aggcaggaag 9420 gcgaggcagt gttgccggatgtgccagtgt ttcaaaagaa gccagagact ccatttttat 9480 ttttttgtat ggaatctcctgattttgaaa tattggcaga taattcaaat tatcttaaac 9540 actacaggcc aaacaaaacatatctgtggg ctagagacag tctgccagtt tgtaactatt 9600 tctccagatc atgagtaaatttggctttac gatggtcact cagttcttat tactctaggt 9660 tgttcaaatg aattaaaaaagctgaaatta tatgaataaa cccctgggca cacatgaaag 9720 aagtgaaaaa cccattgtttcctattgtag aaacatggaa gcatgtcaga gccagaggat 9780 ccagaggaaa tattctcactagcctcagac cctcaggagt gagggagctt ttcttgttaa 9840 tggccacgct tgtgcagttttccttcccag gtgctggtga aagaaaccca cagtcttgga 9900 atcatggaag tgataccataatgactgtca gttgacgttg ctttaaagaa tgaagccaca 9960 gaattgtgct gttagcatgtcgtgagcagt tagttgagtt ggtggcttgt aatttactct 10020 gtgtggatgt tattgatcaaagcttttcat tattgacagt gtctccatct gctgtttgct 10080 gtttttaggg gaaaccaccctttatgactc aacagcagat gtctcctctt tcccgagaag 10140 ggatattaga tgccctctttgttctctttg aagaatgcag tcagcctgct ctgatgaaga 10200 ttaagcacgt gagcaactttgtccggaagt gtaagtttgg ggaacttttt cttgaaaact 10260 gtcctgagag agaaaaactagaaagatgct tgaggcagaa tgagttactg gttgatagta 10320 gtcggtaaga actctggttctatataagac agatccaggt tcaaattcag gctgcacctc 10380 ttatagctgg gagaccaggtaagttgggct tcttggttgc aagcgacaaa cttaattcaa 10440 agactgaatt taggccaggtgcaatggctc atacctataa tctcagccct ttgggaagct 10500 gaggtgggtg aatcgcttgagcccaggagt tcaagaccag cttgggcaac atggtgaaac 10560 cccatctcta caaaaaatacaaaaattagc tgggtatggt ggcttgcacc cgtggtccca 10620 gctgctgagg aggctgaggtgggaggatca ctggagcccg ggaggttgag gctcaatgag 10680 ctgtgattgt gccattgcactccagtctgg gtgacagagt gagaccctgt gtgaataaaa 10740 gagtgaattt attggctcatgaaactgaga aatccaggaa tgagttaagt tttagcttta 10800 ggcatagcta gttccagagacctcaataat atcccgtggc cctgtcctta tactcactca 10860 gggctgactt tctattaggcagagtaggca cggtgcttag gatctgtgat atttaatttt 10920 aatgaattta attacttttaattaactgaa ttaaatttta atttgtttta aaattatagg 10980 aaaaatgaat ataataatgtataatgattc tggattacat tcatctttat actaatgtag 11040 tcataaaata taatttttgttttttttgga gacagagtct tgccctatta cccaggctgg 11100 attgcagtgg tatatcatggctcactgcag tttcaacctt ctaggctcaa gcaatccttc 11160 caccccagtg gctgggactacaggctcaca ctaccacgcc cagctaattt ttgctttttt 11220 ctctgtagag atagggtcttactatgttac ccaggctggt ttcaaactcc aggcttgaag 11280 cagtcttcct gcctcagcctcccaaagctt tgggattaca ggtgtgagcc accatgcctg 11340 gccccataaa atataatttttgaattcttt tttgttttta atggaggaag gggctgagga 11400 aggcaaaagt acctagggcctatgaagtca tatattggcc ttgccttcac cctgtttctg 11460 actttgcttg acttccatgtgatgaggcag ttggctgtta gtgtcccagt ttcatactct 11520 tacattagtg tttttcaaccagtgggtgat ttgacgtttt cggttgtcag agctagttgg 11580 gggtggtggt gtgtgagtttggggggaagg gtcctactgt cagttaatgg gtgaggccag 11640 agatgccacc aaacaccttacagtgcacaa agcagccccc ataacacaga attatgtagc 11700 ccacaatgcc aacagtgctgaatttgagaa accccacctt gtacaacatt gctgtgcaac 11760 caaccaccct aaatattactgacttaaaac aatagtcact gtggctgggc gcggtggctc 11820 atgcgtgtaa gcccagcgctttgggaggct gaggcggcgg atcacttgag gtcaggagtt 11880 ccagaccagc ctggccaacatggtgaaacc ttgtctctac taaaaataca agaattagct 11940 gaatgtggca gcgggcgcctgtaatcccag ccatttggga ggcagaggca ggagaatcgc 12000 ttgaacctgg gaggtggaggttgcagtgag ccaagatctc accattgcac tccagcttgg 12060 gcaatgagtg agactctgtcttaaaaaaaa aaaaaagtta ttgtattacc tcttgtgtgt 12120 gtaggttaat tggactcagctggggattcc tctgctctgt attacattgg ccaggattgc 12180 agtcacctgg ggctctcctgggctggaatg tgtgagaggg cttactcagt gtttggtgcc 12240 ctggcttgga ggctgggcccagctgggcct ctctctcttc atgaagtttc agggcctttt 12300 gctgtccaca tggcacctctatgtggtctc caaatcagaa gtcaaggaac tacagcctgt 12360 gatgcctatt ttgtaaagaaggttttactg gaacacagcc ctacccatgt gtttgtacag 12420 tgcctatggc tgctttcacatcataacagc attttatttc attttattta tttttttttg 12480 agacaaagtc tcactctggctggagtgcag cagcacaatc atagctcact gcagcctcca 12540 actcttgggc tcaagcaatcctcctgtctc agcctcctca gtagctagta ctacaggccc 12600 atgccaccac taatggctaattttttaatt ttgtgtagag atgggacctt gtgagattgc 12660 ctaggctggt cttgaactcctggcctcaag aaatcctccc accttggcct cccaaaatgc 12720 ttggattaca ggcatgagccactgtgccca gcccacaaca gcatttgagt agttgtgata 12780 gagaccaaat ggcctacaaagcccaaaata gttcctgttt ggcccatttc gaaaaggctt 12840 gctgacctct gagctacatggtctctctag caggacagcc tcgacggtag ctcaggtttc 12900 caaaacacaa aagtggaagctgccaggctt tcttaggggt tatcctagga gggacatagg 12960 atctctttga ctgcattttattgtttgatg catgctctgg ggctgctcaa attccacctg 13020 agaggaaact acacaaggtcatgaatccca agaggactgg ggcattgggt gctatttttg 13080 gagactggct accacaccctgcccaatggt aatcttccct tatctagatt aatacaaccc 13140 cagggaagat tctaacttggctctgctttg ggtcatttgc ctccctggag gtgaggtgtt 13200 gtgatcggtt ttgttggaatgcccaaaggg gtcagggcag tgtgattacc aggacctcat 13260 ggaatggggg atgcgtggttatgcaaagga gccggggatg ctgggtagaa aaaaaatcag 13320 catatgttca ctatagtgctcttcagtatt ttacatgtac tttgttctca gttttctcat 13380 ctgtaaaata ggaataatgtatatcctttt tttttttttt tttttggagt cttgctctgt 13440 tgtccaggct ggagtacagtggcacaatct cagctcactg caacctccgc atcccgggtt 13500 caagtgattc tcctgcctcagcctcctcag tagctgggac tacaggcgtg caccaccaca 13560 ctcagctagt ttttgtatttttagtagaga tggggtttcg ccatgttggc caggctggtc 13620 tcaaactcct gacctcaagtgatctgcctg cctcggcctc cgaaagtgct ggaattacag 13680 gcatgagcca ccacgcccattgggaataat gtatatctaa tgaggctgtg ttggaattga 13740 atgagttaat gcacagaccagatttgtcat gttgcctggc ccataggaga caataaatgg 13800 tacccagtat taataactgtgaatgtcaac aacatttaat atattgtata tcttcaaaat 13860 gtacttgagg tatttgttcatcattctgtt tttgtttgaa taagctcgtg ccttcttttt 13920 gtgaatattt aaatttataagtagcgagtg ggaggggaag gaagttatgt gatgaggcta 13980 gcttactgag ccatctgcaggcaccttcat tagtcttgag actgtcctct ggttacttaa 14040 cagcagtgaa ttatctagaatcatttagtg atcagaagac ttggtttagt ggaatgtaga 14100 tttttttcta atagacccctcttccaggga aatgtttcat atttttgaag aggtttcctg 14160 gggagtgttt aagaggccatgattgaaaat gggtgattac attagtgtgt tttctattcc 14220 tccccttttt gagtttctgttttggaatgt aagctttgtt tttctacgtg gagaagggtc 14280 cctcagctgc ttctgcccaggttttttgaa tcttcctata gggatggaga ttttctttgg 14340 ggactgttag agaaaatggaatagagtgta gctctgaagg agaaggatgt ctccagcaga 14400 agtacctcta gccttgggccaagggaggga agggaaggga acgagcatct gggaaccagg 14460 gaagggattt ttgtctttcttaattactct tacatcccca gtgcccaaaa tagtgtctgg 14520 catatgttaa gtccttagtaaatacttgtt gaatgagtgt atgctcagtg aacaaaataa 14580 atggcaaaca ttaagcacagtatcagataa tttgtgtaaa aaatatacag cagtgttata 14640 ctaaaacttg cacagaggccaggtgcagtg gctcacgcct gtaatcccag cactgggagg 14700 ccgaggtggg cagatctttgagctcaggag tttgagacca acctgggcaa catgctgaaa 14760 ccctgtctat acaaaaaatacaaaaagtag ctggggcatg gggacgcaca tctgtggtcc 14820 cagctacttg ggaggctgaggctggagaat tgcttgaagc tgggaggtgg aggttgcagt 14880 aagccaagat tgtgccactgcaccccagcc tgggtgacag agtaagaccc tgtctcaaaa 14940 cacaaaacaa cacccccttcaaaaaaaatc caaaaccacc accacaacaa aaaaacttac 15000 acagaaaagt gttgataattgtcaaaattg ggctgttatt ggcaatttga cagtagctga 15060 attactacca tttgagctatattcactata gataagatct tcaatatatt tacaacttta 15120 gtactaatgg gaaaatgataacttttgaaa agtttttttt ttttcttatt gcaaacaata 15180 cacaatacaa tgttaaatatagaaggttaa acgtgcatct gagtctgttt gggctgcgat 15240 aatagatacc ttagacttggcaatttataa acaatagaaa ttcattgctg acagttgtga 15300 agactgggaa gtccaagatcaaggcgccag cgaatctggt atctggtgat ggctccctgc 15360 ttcaaaaatg gcgccttcttgctgcatctt cacctggcag aaggggcaaa catgagtcct 15420 tcagcttctt tttttttttttttctatgtt taaaactttt ggtccggcgt ggtggctcat 15480 gcctgtaatc ctagcactttgggaggccga ggcaggtgca tcatgaggtc aagagatcga 15540 gaccatcctg gccaacatggtgaaaccccc ccgtctctat actaaaaata caaaaattag 15600 ccaggcatgg tggcgtgtgcttgtagtccc agctactcag gaggctgagg caggagaatt 15660 gcttgaacct gggaggcagaggttgcagtg agccaagatt gcgccactgc actccagcct 15720 ggcaacagag taagactccgtctcaaaaca aacaaacaaa aaaaacaaaa aaaaactttt 15780 attttaggtt catgggtaaatgtacaggtt tgttatgtag gtaaacttgt cttggggttt 15840 gttatagatt atttcgtcacccaggtacta agcctagtaa ccaatagtta ttttttcaga 15900 ttgtctccct cctcccaccctctgtcctct agtaggctcc aatgtctgtt gttcccttct 15960 tagtgtcctt gtgttctcatcctttagctc ccatttatat gtgagaacat gtggtatttg 16020 gttttctgtt cctgcattagtttgctaagg ataatgtcag cctctttttt tttttttttt 16080 ttttttttga tacagagtctcgctctgttg cccaggttgg agtgcagtgg tgcgatcttg 16140 gctcactgca acctctgcctcccgggttca agtgattctc ttgccttagc ctcctgagta 16200 gctgggacta caggtgcgcaccaccatgcc aggctaattt ttgtatttta gtagagatag 16260 ggtttcacca tgctggccacgctggtctcc aactcttgac cttgtgatcc gccggcctcg 16320 tctttttccc aaagtgctgagattacaggt gtgagtcact gcacccggcc caatgtcagc 16380 ctctttttta gggaagtgatttaatcactt ccctaaaagt cctacctcgt tttttttttt 16440 ggttttttct tttttttttttttttttttt tttttttttt taggtagagt cttgctctgt 16500 cacccaggct ggagtgcagtggtgcgatct tggctcactg caacctccac ctcctgagtt 16560 caagcaattc tcctgcctcagcctcctgag tagctgggat tataggtgcc tgccaccacg 16620 cctggctaat ttttttgtatttttagtaga gttggggttt caccatgttg gccaggctgg 16680 tcttgaactc ctgacctcaagtgatctgcc caaaatgctg ggattacagg cgggagccac 16740 tgtggccagc ccctgcaagtcctacctctt aatagtatta cactggggat tacatttcaa 16800 catgaatttt gtaggggcgaggggcacaaa cgtttagaat atagcacatc acatacatag 16860 tgagagaaaa atccctcaaaatcttacctg agacaatcac tgccaacaga ttgctgtata 16920 gtgtgccaat tttgtttgtgtgtgtgtgtg ccttaaaaat atttattatg gaaatttaaa 16980 aacgtacccc aaggtggccaggtgtagtgg ctcacgcctg taatcctggc actttgggag 17040 cccgaggtgg gtgtattacttgaggtcagg agtttgagac cagcctggcc aaaatggtga 17100 taccagtctc ctaaaaatacaaaaattagc cgggtgtggt gggcacctgt agttccagct 17160 actcgggaga ccaagtcatgagaattgctt gaaccctgga ggcagaggtt gcagtgagcc 17220 aagaccatgc cactgcactccagccagggt gacagagtga gactccatcc tagaaacaaa 17280 caaacaaaca aacaaaccaactaaccaacc agagaaaact ccctgtctgt aaggagtatg 17340 tgttctaatg gatactgagccatcttgttc tgtttaacat gtgcctaatg ttcttttata 17400 tgggcggact tgtaggttgtttcaactttt ctgttgatga acctttaggt ggtttctgat 17460 tatttttgtg ttacaacagttttcatcatt cacatctttg tatgcatctt ttttgagcac 17520 atgtgcaagt atttctgtggacaatggatg attcctagaa attgaaagtt tggattactg 17580 tgttccaaaa aaggaagcaatacacccagc tatgttggct tttgctcttg ggtccagatg 17640 attatctgac aaagttattctctgattgca ttttcttttc ttttcttttc tttttttttt 17700 ttgagatgga gtttcgctcttgttgcccag gttggagtgc aatggcgcga tctcggctca 17760 ctgcaacctc tgcctcccaggttcaagcga ttctcctgcc tcagcctcct aagtagctgg 17820 cattgcaggc atgcgccacgacacctggct aattttttgt atttttagta gagatgggat 17880 ttctccatat tggtcaggctggtcttgaac tcttgacctc aggtgatcca cccgcttcag 17940 cctcccaaag tgctgggattacaggcgtga gccacagtgc ctggccctct gactgcattt 18000 tcacagtgtt ttgggtccttatctctacct cagtacctca atattcagtg cccactgggc 18060 ccttagatac tgcagctaaaagtgcacagg ggtggagtga tgtgacggtt ttggggtcac 18120 agaagcagct ggtatagagagaagttgtga agtttttttt ttttttcctg agacagagtc 18180 tcgctgtatc ccctaggctggagtgcagtg gcttgatctc ggctcactgc aacctctgtc 18240 tccctggttc aagtgattcttatgcctcag cctcccgagt agctgggatt ataggcatgt 18300 gtcaccatac ccagctaatttttgtgtttt tagtagagat ggggtttcac catgttggcc 18360 aggctggtct tgagctcctgacctcaggtg atccgcccac ctgggcctcc caaagtgctg 18420 ggattacagg cctgagccattgcgcctggt cttttttttt tttttttaag taatcatagg 18480 cttgaatgta gcctctcatctgttcacctt aataatccaa aagcctttag ataaagaaat 18540 ggagatttgg aatggcttctcagaattcca agagagtatt gtcatggttt tgcctgcaaa 18600 gcaccgtggt ctgtctccttgtgcagttga gaaagctggt ggtcgccact gacaggccca 18660 gagttattaa gttggacactgctttaagca actttgtaaa caatccaagg catactagag 18720 aattaggaga gattggctttgtgtatgagc aataacaaaa tcaagttcaa tccagcaagt 18780 ttttggggaa ttataattcaaaactcaaat acttgatctg gaagaaactt ggaaagaggg 18840 aaggaagaca ggcttgttacagcattgtca gggtaaaagg aaaataccgt gcagctttta 18900 attttgcttc ttcatggcattccccatgta ggtgccctag atttgttttt tacagtggtc 18960 acgacttcat gtggatccacccaccactct tgcctggttc cccaagggac caagggaagg 19020 tgtattcagg atgattgctgaagtgagggg tggggtctgt ggctgagaag actctcaata 19080 ccgcggcact cattataagcctctgacaca ggagatttca actccacccg tgcaacaaag 19140 gaacagggtg ggcaagagtagttacagttg caggctgagt gcgatggttc atgcctgtaa 19200 tcccagtgct ttgggaagccaaggtgggag gattgcttga gtctaggagt ttgagaccag 19260 cctgggtgac ataatgagaccctacctgta caaaaaaatt ttaaaaatta gccagattgg 19320 tggtgtgcgc ctatagtcccagctactctg gagaatgagg tgggtgaggg tcccttgagt 19380 ccaggagttc gaggctgcagtgagttatga ttctatgatt tcaccactgc attccagcct 19440 gggcgacaga gcaagattgtgttctttttt ttttttgaga cggagtctca ctctgtcacc 19500 caggctgaag tgcagtggtacgatctctgc tcactacaac ctgcacctcc caggttcaag 19560 tgattctctc cctcagcctcccgagcagct gagattaaaa gcggccgctt gtgtgcagct 19620 aatttttgta ttgttagtagagatggggtt tcatcatgtt ggtcaggctt gtcttgaact 19680 cctgacctca ggtgatccacccgcctcgcc ctcccaaaat gctgggatta caggcgtgag 19740 ctactgcgcc cagccatttgtgtctcttaa aaaaaaaact aagaaaatga aaaaaatgac 19800 attggccaat tcattaaaatgccactcact gactgtggta tgaaatggct ttccctttga 19860 tggaccgagt ctgtctcattgtgtgagcca cttgcagggc tgagtatgac tctggaatgt 19920 agctcctaac cttatctgctgcccagccat tgaaatggcc atcccttcca gttcccagaa 19980 gattccagtg tgtgtttgggattttaagac agtctcttgg tcttcagtgt ggcatctttc 20040 tgccggattt tccaggataattttgattat aagcattgca ttgcccttgg tgtgtaatgc 20100 ctgtgtatga tgctgttcccttgtaacgtg caggattaaa tttttgggtc agccactgct 20160 gctccccttc attcctgcaggtcattagag tcatcgtaca tttagcgatg tctcagatca 20220 gtgtatctag agtgttaataaacatgttag attccaaatc tactgtccat ttaatccata 20280 cttcatacgt tgaggatctctgactgaaag attagacttg gaaaaataat aagactgtat 20340 ggtaagaaaa ctatagttgcaaatccattt ggacatgtag tatgtcagcc ctgcagagca 20400 gatgtcagaa ccccatttagttctctgagt gctaagccct tctgcccacc acgctgtttt 20460 ttttttttga gatggagtctcgctctgtca ctcaggctgg agtgcagtgg tgtgatctcg 20520 gctcactgca agctctgtctcccaggttca cgccattctc ctgcctcagc ctcccaagta 20580 gctgggacta caggtgctcaccaccatgcc cagctaattt tttgtatgtt tttggtagag 20640 acggggtttc actgtgttagccaggatggt ctggatctcc tgaccttgtg atccacccgc 20700 ttcggcctcc caaagtgctgggattacagg cgtgagccac tgctcctggc ccccacgcct 20760 tttttttttt ttggagacagagtttcactc tgtcacccag attggagtgc tgtggcacaa 20820 tctcagctca ttgtgtcctctgcctcccag gttcaagtga ttcttgtgcc tcagcctcct 20880 gagtaggtgg aattacaggcgtgcaccaca acacctggct aatttttgta tttttagtag 20940 agatggggtt tcaccatgttggccaggctg gtctcgatct cctgacctcc agtgatccac 21000 ttgcctaggc ctcccaaagtgttgggatta caggcgtcag ccaccatgcc tggacccctc 21060 tgccccttta agcactgccacatattagat ctacgaaggc tttatggata caatccaagg 21120 aagatgaacc ttgggctagtgggataaaac taagcgcatg tagttagaat ggaatgatct 21180 ggaaaccagg tcccaagttggtctaaatta gactcatgtt gactatgtca cactgtaaac 21240 cagtctaaat gctaataagcatgcttgacc aaacactgcc ctgcagcctt cagagaggaa 21300 gaaggaaaac ataatttgtatcctctctcc ctattttctg agtctatggg attcaaattg 21360 tagctgccat ggaaactgtactttggaatt tctagagccc ttaattttaa cttaacatat 21420 aaaaacactt ttgtactgattttataatta ttcatgatgg atgagaaagt gaatgtcttt 21480 gacagtgagg gaagctatccgaatgctatt ttcttttttt tttttctttc ataaagatgc 21540 atatatttgc atgctttatttacctggggc taactcttgc atcttttgca gattccgaca 21600 ccatagctga gttacaggagctccagcctt cggcaaagga cttcgaagtc agaagtcttg 21660 taggttgtgg tcactttgctgaagtgcagg tggtaagaga gaaagcaacc ggggacatct 21720 atgctatgaa agtgatgaagaagaaggctt tattggccca ggagcaggta ggaggatttt 21780 aacatcatgc ttttccactttctgtaccgg agtgttcatt gcaaagacga taatctgctg 21840 cactggcgtc taggatcaagcacgttttcc tctgtgactc tatatttaat tatagttggg 21900 gcaaaaaggt ctctcatgttcttagctcat cttcttgaac tgatgttggc taattttgaa 21960 ggctcacaaa ttcctcttgatgtatcatgt ttctatcgtt gtaatttatt tcagaaccaa 22020 ggtggccttt tagctaatgaatttaagatg atcttttatg accattagct gaggactcag 22080 gatatacata tggtggggtgaatcagattg cttttgtaca cgctttaggt atttgtgttg 22140 tgggcatatg gatttggttttaaaacaggc ctttgaagaa atcaaataac attctttgtt 22200 atgtggctag ggagttgcttgtttgagagc aggtagaacg ttatcttttt tgttgtggta 22260 tttttctttc ttttaaacaaggctactgtc tctagacata ttgattcatt tgctgtgttt 22320 tagagagatg gccgtcagccttggaattca gagagtaatt tattacttac agacatttta 22380 gtgcacatga tatgtctgataatgtaccca gctctgcagg aagcttgcaa aaggaataga 22440 agtcccatgg ttgctattttcagtgtttaa aaacaacctt ggaaagtgga ggaaaaatgc 22500 aaatgtataa agcaggtgcttaccagctaa agtatcacag aagtgggaga gcaattagca 22560 aattaattaa cgatgatgtgaggggagatg ttgtgggtga gcaagggaca gttagggaca 22620 gttctcaccg atggggggaaatgtaggttc tcggcagaga gaagtgatga gaacatgttg 22680 ggtagaagtg tgacattctggagtactaga atgctatgca agtgtgtgtg tgtgggtgtg 22740 tgtgtgtgtt cagtggttcagaacagactg ggaaatggcg aaatgaggac atttgggtgg 22800 ggagggggaa atgggtgggaaactcaagaa ccttttttta aaaaattgtg gtaaaatata 22860 tataacataa agtgtaccattttaaccatt tttaaatgtg caactgagtg gtattcagtg 22920 cattcatgat gttgtacaaccatgaccgct ctccatttct agaatttttc tatcatccca 22980 aacagaaact ctctatccattatacaatac ctccccattc ccccaagaac cagtttttga 23040 attgcagttt actttgtgaggctgttgggg attatttagg cctctggaag gaggaggttg 23100 ggatcagagt ctggccctgtggacttcaat gactttgtgt ggcctccaat cagagaagca 23160 gcggagggca ggaagctgcttgtcagaatc tgagagtgat gtggcttctt tgtttagcaa 23220 taaaatgtga gcacataatagaaaggaaaa gtgacaggac atggcagata atttggaaga 23280 gaggagtgga agatgctcactcagcctccc agctcctgag aaagaactgt gtctcatcag 23340 ttcatactac ctgagcatctgttgtatctg gtgtgtttct aggtcctgga gaagaggcat 23400 tacgtgtagc cctgaccttgtgatgcttat gtttttgatg ggaaatagtg cgtgtaaaaa 23460 gaaaataatc caacaggccacacggcaggc aaacaataga gatattcaaa taggtatacc 23520 ttcctccagg tgaatggcctgaaatgaccg tgtggaagtg tgggctgggg gcttataaaa 23580 ttatacacat acaggcgctaactaaagccg cctattcatt ccttaagagg atgcatagaa 23640 aagaaaagta gggtccttaactgagccatt tggaatttaa gggcatgaga gaagccagca 23700 caagcagtga agggaaggaaaagaagtgcc cgagaggagg gagggatgct gttctgcaga 23760 caaggcctgc cgcctgggagaggcccgcac gcccacccag ggttctctga cagctggaag 23820 gggtcttcag agactgtttatattttattt atttatttat ttatttattt tgagacagag 23880 tctctgtcac ccaggctggagtgcagtggt gcgatctcag ctcactgcaa gctccgcctc 23940 ccaggttcac accattctcctatctcagcc tcccgagtag ctgggactac aggcgcctgc 24000 cacaatgccc ggctaatttttttgtaattt tagtagagac ggggttttac ctcgttagcc 24060 aggatggtct tgatctcctgacctcatgat tcgcccacct cggcctccca aagtgctggg 24120 attacaggtg tgagccactgtgcctggccg actgtttcta ctattttaga gagagggtct 24180 cactgtcatc tgtgctggaatgcagtgatg cagtcatagc tcactgcacc ctcaaactcc 24240 tgggcttaag cgaccctcccgcctcagcct cttaagtagc tgggaccata ggcatgtgct 24300 gccacaccca gttaactttattatttattt atttatttag agaatgagtc tcattctgtt 24360 gcccaggcta gaggtgcagtggcacgatct cggctcactg caaccccgcc tcccaggttc 24420 aagcgattct tcttgctcagcctcctgaat agctgggatt acaggcacct gccaccacac 24480 ctggctaatt tttgtatttttagtgcagag ggggggtttc accatgttgg tcaggctggt 24540 ctcgaactcc tgaccttgtgatctgcctgc ctcggcctcc caaagtgctg ggattacagg 24600 cgtgagccac cgtgcccggcccactttatt attttaaaaa cattgtttta tttttatttt 24660 tttgagacag agtccgctggagttcagtgg ccggatctca ctcactgcaa cctctgcctc 24720 ctgggttcaa gtgattcttgtgcttcagcc tctctagtag ctgggactac aggcgggtgc 24780 caccatgcct ggctaatgttttttgtatct ttttagtaga gacggggttt tgccatgttg 24840 gccaggctgg tctcgaactcctgacctcaa gtgatctgcc cactttagcc tctcaaagta 24900 ctgggattac aggcgtgagccactgtggct agcccccagc taactttaaa aaaaaatttt 24960 gtgggccggg tgcagtggctcacgcctgta atcccagcac tttggaggcc aagcagggcg 25020 gatcacttga ggtcgggagtttgagaccag cctgaccaac atggagaaac cctgtctcta 25080 ctaaaaatac aaaaaattagccgggtgtgg tggtgcatgc ctgtaatccc agctacttgg 25140 gagctgaggc aggagaattgcttgaatctg ggaggcagag gttgcagtga gcttagatca 25200 cgccactgca ctacagcctgggcaacaaga gcgaacactc cgtctcaaaa aaaaaaaata 25260 aattatgtag aggtgggatctccctatgtt gcccggactg gtcttgaact cctggcctca 25320 agtgatcctt ccatctccccctcccaaagt gttgggatta caggcatgag ccacccctcc 25380 tggctgagac tgcttattttatttattttt aatttttttt gttttgagac tgcttatttt 25440 aatggaagct tcaggggtcagacggggtca gacagagtca ttggtgagca agcaaaggtg 25500 tagactgttc agttcagccttccttggaca ccttttatgt gccagacaaa agaaggatca 25560 gcatatcagg tgcagtaaattattggggtt atgttggtgt ttcccaaatg tgttagattt 25620 atccctggta gtgttaaatctcatgatttt aggtagtata tggacaacct atgtaaaaac 25680 atttaatagt ttaatattaactagcatatc aaaacctgtg actttgctca cgcctgtaat 25740 cccagcactt tgggaggccaaggcgggagg atggtttggg cccaggagtt tgaggccagc 25800 ctaggtaaca tggtgagaccctgtctctaa aacaaaacaa aacaaaacaa acaaacaaac 25860 aaataaacaa atcccctgtaacttgttcta acaataacct aaacaatttt ttatttaaaa 25920 ttaaataaaa aaattgaaacagtaaccatt tttttttttt tttttggaga cagagtcttg 25980 ctttgtcacc tagtctagagtgcagtggca caatctctgc tcactgcaac ctctgccttc 26040 aaacaattct cctgcctcaggcttctgagt aggtgggatt gattacaggt gcactccacc 26100 atgcccagct aatttttgtatttttagtag agacggggtt tcaccatgtt ggctaggcta 26160 gtcttgaact cctgacctgcagtagtccac gtgccttggc ctcccaaagt gctgggatta 26220 caatcacaaa tttatagaaaagttgcaagt accatgtagt cagggttctt aagagaaatg 26280 gaaccagtag gagatagatatataatcatc tcctaggatt ataagttgac acataagact 26340 aaccgtcaca tacagtataaacaacttttt ttcttaaacc atttgataga tacacacaca 26400 ctgatataca tagaatatatatacacacac acagaatgta tatacacata gaatatatgt 26460 gcatacagaa tatatacacagaaatatata tgtacacatg catagaatat atttacatat 26520 atatgcatat atataatttatttattttaa gcagttgatt tatacagttt ttgtttttgt 26580 tttttttttg agacagagtctcactctgtc acccaggcta gagtgcagtg gcgagatctc 26640 agctcactgc aacctctgcccccgggttcc agtgattctc ctgcctcagc tccacaagta 26700 gcacaccacc atgcccagctaatttttgta ttttttttag tagagacgag gtttcatcat 26760 gttggccagg ctggtctcgaactcctgacc tcaagtgatc cgcccgcctt ggcctcccaa 26820 agtgctggga tttcaggcgtgagccaccac acctggctcc cataatgtct tttagaataa 26880 aacgatcgag ttgaggatcacacgtgacac ttaattgtcc tgtctcttta gtctccttca 26940 atctggagca gttctttgatttttcctgga ctctcatgac cttgacaatt ctgatgatta 27000 taggccagtt attttgtaaaatttgaattt gtctgatgtt gcttatgttt agatttaggg 27060 tcttggtctt tggccggaatatctcagaca agatgctctg ttcttattgc atcagagcag 27120 aagactctct gtttcagttgatcacattta tgttgatgct cactttgatc acttgattaa 27180 ggtggtgtca gttatgcctttctacttgta gggttactcc ttcctccttc gtgattttat 27240 ttattttatt tttcttagagacagggtctt gcttggttgc ccaagctgga gtgcagtggt 27300 gggatcttgg ctcactgcagccttgaactc ctgggctcaa gtaatccacc tgccacagcc 27360 tcctgagtaa ctgggactgtaagcgaacac caccacaccc agctactttt tgtattgtag 27420 agatggggtc tcactgtgttgtccaggctg gtctgtaact cctggcctca agcagtcttc 27480 cggccttggc ctcccgaagtgctgggatta caggcatgag ccactgcacc cagcctcctt 27540 tgtaattaaa aaagtattttatggggagtt actttcaagt gatggaaata ttttatatct 27600 atgtggactt ggattttcctatttcagtca gtgagttata atccatttct gtcactagtt 27660 ttatacttaa attgttcccaacttggccac tgagaacctt tttaggttag cttttgtgtc 27720 cttttcacat gtctccaagattcattgaat actttcctgc tttctggtat agcaagatgt 27780 tcaggttctt ttggtacttttactttctct gccctggctc tggcatcagt catttctcag 27840 aggagccctg tgcctttcagtggacaatgg tgtttagagg ccaagatctg gacattgggt 27900 gttttcattg ctaccggtgtgtcactactc ccagacccct ttcagtggac agcactaagg 27960 aatacacata cgtatatacaatatatccac ctacacatgt gcgtgcactc acacacacac 28020 atatacatta catctatatttgtgtatcca tgtctatata ttgaaaattg tggctgggca 28080 cagtggctta tgcctttaatctcagcattt tgggaggctg aggcaagagg atcacctgaa 28140 gccaggagtt caacaccagcttgggaaaca gagagagact ctgtctctac aaaaataaaa 28200 agggaaaacc atgagttcacacccgtgccc ccagttccaa tccaacttca cagggttcat 28260 tttagttttc accctttccatgtttgtaat tctcttctct gacattatac ccttaatatg 28320 tttacttatt ttatgcatctgtatgcatcc aatctactgt ctttgttggt atcccacctc 28380 cccttggtgg gtccagataatctgctctgg gttgcccttt cacgtggatg tcttccttac 28440 cctgtgtggg cctgtgatactgggctgccc ccacacatga gtgctgccct cctcacgttg 28500 cttgggacgg cactgtgtcctgggccacca tgacttttct cataactagc gtggatgctt 28560 accttgttcc acaccagtgaatggcttcag gaagagaaga ggaagagaaa aatatttaca 28620 tttaaagaaa ggtagtttaaagaaatatgt taggtaaaga attgagcagg taatatacgg 28680 agctggcaaa aattgtgaccaaagtaggtg aatgattgag atttatgcaa ttctgggcta 28740 agtgacagcc ccttccctttcccttccctt ccccttccct tcccttttct tccctttccc 28800 ttccctttcc ttccctttcccttccccttc ccttcccttt ccttcccttt ccctcttctt 28860 ccttccttcc ttctgttttcttttcccttc tttcctttgc cttttttttt tttttaaagc 28920 tagaaacatc agtttaggcataaagacaga ggaaaaggct tctttttcct ctcacagttc 28980 tttataattg tctaagcagtttcttttttc cctaggtttc attttttgag gaagagcgga 29040 acatattatc tcgaagcacaagcccgtgga tcccccaatt acagtatgcc tttcaggaca 29100 aaaatcacct ttatctggtgagtctttaca tctgtctctc tggaattagc ctagcactct 29160 gacactcaga tgcctgtggtagaactgaat gttgttcttg cccatgtggt ctcattcatg 29220 caaagacttt cttaccttacaggtgtctcc ctggtttcct cgttataaag atcaagagct 29280 aacccattta gaaacagcctcattgggctg aacgtggtgg ctcacgcctg taatcccagc 29340 attttgggag gccgaggcgggtggatcacg aggtcaggag atcaagacca tcctggctaa 29400 cacagtgaaa ccccgtctctactaaaaata cagaaaaatt agccgggcat ggtgtcgggt 29460 gcctgtagtc ccagctactcaggtggctaa ggcaggacaa tcgcttgaac ctgggaagcg 29520 gagcttgcag tgagccgagattgcgccact gcactccagc ctgggtgaca gagcaagact 29580 ctatctcaaa aaaaaaaaaaagaaaaaaaa agaaacagcc tcattgacag ttggatattg 29640 tagctgtggc tttcaggcaataatagggaa tcatttattg gggaatagtc tgtcattatg 29700 tataagataa tcttgctttaatttttaaaa acttcctgtg ttagcttgct taggattaaa 29760 aaaatgataa tagtgcatggttgttataag aaaatgcaaa cactgcagac atgcatgaag 29820 ttgaagggaa agccccccattttcttttcc ttttcttttt ttttgagaca gagtctcgct 29880 ttgtcaccca ggctggagtgcggtggcact atctcggctc actgcaatct ccacctccca 29940 ggttcaagag attcttctgcctcagcttcc ctagtagctg ggattacagg cacgtgtcac 30000 cacgcccaac taatttttgtatttttagta gagatggggt tttaccacgt tggccgggct 30060 ggccgcaaac tcctgacctcaaatgatcca cctgcctcgg cctcccaaag tgttgtgatt 30120 acaggagtga gccactgtgcccggcctctc cgttttattt tctaatcctc ctccctaggg 30180 gaagaaatgt taaatggttacataagcttt ccctttctga cccttaactg tgctctgtag 30240 gagcatggtg ggggatgtttcttttctttt cttctttttt tgagaccagg tctcactttg 30300 ccacccaggc tggagttcagtggcatgaac atggctcact gcagcctcga cttcctgggc 30360 tccagcaaac ctcccacctcagcctcccgg gcatacacca ctgtgcctgg ctaatttttg 30420 tatttttagt agagacggggttttgccatg ttgcccaggc tggtttcgaa gtcctgagct 30480 caagagatct tcctgccttggccttccaaa gtgctgggat tacaggtgtg agccaccatg 30540 cccagctccg gtgggggatatttctatatc cacatgtgta tagtttactt tataaaaatg 30600 gtatgttact ctgtgcttggctctccagct tgctgttgcc tttcaccagt gtatcccaga 30660 catcctttct tccttgtcagtaacgcaggt ctactttatt ctttgagcag tggcataatt 30720 ttccctgatg tgtatatatcataagttaga gaatgctaaa attcattttg gggccttgtt 30780 taggttcttg agggattaaattcctaaatt taacaagtgt atcctggaaa caatttttgt 30840 tcctgattca gcccttaaaagaggactatc atgttacctt gaatggagat aaacaggctc 30900 acgtaagaga aaagggtaagagggatgaac tcccacttat cttaaacttc tactggcccg 30960 tttttgggga atttgctgcttttattcctg acctaaaata aataagttta tgtgtcttgg 31020 tttcatatta gttgagaacccagtgcctgg agagaagttt tccttgtcct ctgagtgagg 31080 acattcacat atgaatctattggcagactg gctttgactg accacacgtg ccttcagaac 31140 caatgccaca gctcttaggtttatggcctg aaacaccctt tccttacata ttgccttaga 31200 aactttcctt ccttgagacatggggcatgg aaccctcacc ttcacagatg accttggtgt 31260 gtttctaggg ttgctggtgttccaggacat ctgttgcaga tgcagtattt accttgtgct 31320 ctctgcatca taagcagcttctcatgtttg aatgtattaa cagactttta atttttttta 31380 tttttgagac aaagtctcactctgtcaccc aggctagtgt tacccaggct ggagtgcaat 31440 ggctcaatct cagctcactgcaacctccac ctcctgggtt caagcgattc tcttgcctca 31500 gcctcccgag tagctgggattacaggtgca tgacaccacg ccctgctaat ttttgtattt 31560 ttagtagaga cggggtttcgccatgttggt ggggctggtc tcaaactcct gacctcagat 31620 gatctgcccg ccttggcctcccaaagtgct gggattacag gcgtgagcca ctgcgccttt 31680 tcttttcatt ttttttctgagatggagtct ttctctgtca ccaggctgga gtacagtcat 31740 gcaatctcag ctcactgcaacttccacctc ctgggttaaa gtgattctcc tgtcttagcc 31800 tcctgtgtag ctgggactacaggcgtgtgc caccgtgccc agctaatttt tatattttta 31860 gtagagacgg ggttttgccatgtgggttag gctggtcttg aactcctgac ctcaggtgat 31920 ccacccgtct tggcctcccaaagtgctggg gttataggcg tgagccactg tgcccagcct 31980 caggcttctt tattaagaagaagttcgggc caggtgtggt ggcttacacc tgtaatccca 32040 gcaatttggg aggccgaggtgggcagatca ggaggtcagg agatcgagac catcctggct 32100 aacatggtga aacctcgtctctactaaaaa tataaaaaat taggcaggta tggtggcggg 32160 tgcctgtagt cccagctactcgggaggctg agggaggaga acggtgtgaa cctgggaggc 32220 ggagcttgca gtgagcccagattgtgccag tgcactccag cctgggtgac agagcgaggc 32280 tccgtctcaa gaaaaaaaaaaaagacgttc ccttgaaaca acagggcttt tgtttgtttt 32340 ggtttgtgtt tgtttgttattgttgtttta gatacgtatt tttttctttc tttttttttt 32400 ttaagtgatg atgtctctgttgcagtggca tgatcatagc tcactgtaac ctcaaattgc 32460 agggctcaag tgattctcctgcttcacctt cctgattagc tgggacaaca ggtacaaacc 32520 accatgccta gcgaatttttaaatttttca tagagactag ggtctcacta tgttgcctag 32580 gctggtttcg aactcctggccccaagtcat cctcctgcct tggcttccca aattgttggg 32640 atcacaggca tgaatcaccacacccagcct atttttagat attttaattc gagctctaca 32700 ggaggtttag aacactagcttgtgaagata aacttcattt tcaaggccac acagaatcta 32760 agtggtcctg gaattaggaagggctttgat tttttggacc aaagttgaga gtccacagtt 32820 ttctggtcta ccttgcactgctccataaac tcatatttct tttctctgag ctgaagagct 32880 ccccttcttg gtgtctagtctcaggcaact tattcttaaa agtaagcatt attgaaatgc 32940 tttgggattt tcacatcatcaaggtccatt ttggtagagg cactgacaga ttttgagtgt 33000 tctgtgtgaa ggaactcagttgaggattta gtggtccatg tggcaggcta ctgctcagta 33060 gcttcaggga aaccactgcttgcctcccct gtggccagtg aggatgatca gaggagtccc 33120 agcaggaatg cccaaatgtagttttcttac atgttgatgg gagtgcattg tttcatgtct 33180 aaacagttct caaatcacatcttcaggagg gtactatctg ggcactttga taatttctca 33240 ctttgatgtc accgttcttattaccatcac ctagttttgt catagtagaa ataactttcc 33300 tttttctgtg tgtgtgtgtgtgtgtgtgtg tgtgtgtgtg tgtgtgtttt gagatggagt 33360 cttgccgtgt tgcccaggctgtagtgcagt ggcgtgttct cggctcactg caacctctgc 33420 ctcccgggtt ctcctgcctcagcctcccga gtagttggga ttacaggcgt gtgacaccac 33480 gcccggctca tttttgtattttcagtagag atggggtttc accactttgg ccaggctggt 33540 cttgaactcc tgaccttgtgatccgcccac cttgacctcc caaagtgctg ggattgcagg 33600 tgtgagccac cacgcctggctttttttttt ttttttttga gacagagtct tgctctgttg 33660 cccaggctgg agtgcagtggcgggatcttg gctcactgca gcctccacct cctaggttca 33720 agcaattctt ctgcctcagcctcctgagta gctgggatta caggtgccca ccaccatgtc 33780 cggcaaattt ttgtatttttagtagagaca gggtttcacc atgttggcca ggctggtttc 33840 taactcctga ccccaggtgatccgcctgcc tcagcctccc agagtgatgg aattacaggc 33900 atgagccact gcgcctggccacctttgtct tcttagttgt ggatttaact gctgtggaca 33960 tctgcttggg catagccttcccggagtacc tcttggattg ggactgtctg tgggtttctg 34020 tgctaggaca ggctcccagatgtaggaggc ttccccaatg atctcaccac tggcatcggc 34080 atccttagct tctactcagcttttccatct gccatcttgc aagatggaag gttgttttgt 34140 ttttgttttt gttttttggtttattttttt tgagatagag tctcgctctg ttgccaaggc 34200 tggagttcag tggcgcaatctcggctcagt gcaacctcca cctcctgggt tcaagtgatt 34260 cacctgcctc agcctctggagtagctggga ttacaggcgc gtgccaccat gttcgtttaa 34320 ttttttgtat ttttagtagagacggggttt caccgtgtta gccaggatgg tctcgatctt 34380 ctgacctcat gatccgcctgcttcagcctc ccagagtgct gggattacag gcgtgagcca 34440 ccgtgcccag cctaggagggttcttaatgc agctgttttt tggagttctg gttgcctcag 34500 cacactgcta cttgggtcaatgacattttt actcccttgt tttgtagctc aattgggtat 34560 tactgatggg attttgtaattattaatatt ttcttgtctc cattttcttc tcaagtactt 34620 tgttgctttt gagtaaaatgcttgctaagg gtatagtttt cacataaaag ctcaaattta 34680 gcatggaaat taagatatgctcatacgtct gccatccctt atctgtaatt ctgaaatacc 34740 tagagttctg aataacctcaaattcttttg ttacttgttt atcagcaaaa cctgatttga 34800 actcagtttt tggcaaaacttgatccaagc tctcttaagg ctctttttag tctttattca 34860 ttccctttag tgtgacttcccattttgcta taaaattatg agtgtgtttg attacaaggt 34920 gatgtcccag accctactgagggtgttaca taatataaac tgtatgtatg gctgggcgcg 34980 gtggcttata cctgtaatcccagcactttg ggaggccgag gcgagcggat aaccttagtt 35040 caggagttca agcccagcctggccaacatg gtgaaacccc gtctctacta agaatacaaa 35100 aattagccag gcatgatggtgggcgcctgt aatcccagct actccttagg ctgaggcagg 35160 agaatcactt gaacccaggaggtggaggtt gcagtgagcc aaggtcatgc cactgcactc 35220 cagcctgggc gacaaagcaagaatctgtct caaaaaaaaa aaaaaaaaag tgtgtgtacc 35280 actttacctt tctaaaatctgaaaaattct gaatctggaa acccattctg cttcaagata 35340 aatggatcct agatttatatcggtaccgta cagtcctgaa attccatcct atctattggc 35400 cacttttaca tcaacaaacctttgaagttt ggggaaactt acatatcacg ctcccttggc 35460 agttgaacat tatttatttattttgagatg gagtctcgct ttgcccaggc tggagtgcag 35520 tggcgcgatc ttggctcactgcaacctctg cctcccgggt tcaagcaatt ctcctgcctc 35580 agcctcctga gtagctgggattataggcat gcaacaccat gcccagctaa tttttgtctt 35640 tttagtagag acggggtttcactatgttaa ccaggctgtt ctcgaactcc tgaccttgta 35700 atcttccctc ctcggcctcccaaagtgctg gaattacagg cgtgaaccac cacgcctggc 35760 cctgaagata cattttaaatcaatgaaaaa aacaacagga ttctacctcc tatggtatat 35820 ccctcctggc tgtctcttctctccagtctt gcctctgctg tgtgggtttc aggcatccat 35880 cttctctact ctgaattactgtgataacct ctgaagtatt ttccctgcca tctgtctggc 35940 ccttctccca ggtcttccacatactgcagc caagtcagcc cgctgttgaa acccttcaag 36000 actccctgct gtcctctggatgaagtccag actcttccac gtgacttacc aggcctttct 36060 tgcacttgtc cccagccacttactgtttct ctctttctac cttaacatcc tgaacttcct 36120 ttggttcttt gaccttgcctctgacctttt tccatgctgt tcactctttc cctgttcacc 36180 ttgctaactc ctctttctctttctgggttg gatcagattt cacttcttcc agaagccctt 36240 cctagaccct atacttctggaatggcgcct tttgactgta cgctcattgc accctgtact 36300 tctcctttat gagtgggtgctggtctgtcc cactaggcta cttcatccat aaagggagag 36360 tagagcttta ccaagtcaatgcttaagcaa tatttattgg atgaatgtgt gattaatttc 36420 atagaaattt gatgtgcattcaaatttact tattgtatta cggaacttgc attatattct 36480 cagtggagtt attttctttcacgtgtgtaa ttcaagatag actcagtgag attttcaaaa 36540 tttggaatgc agtgcaaggaaattgaactt gagttctttt gcattttgat ggttaaaaat 36600 ttcccatttg tggtgacataccacaataag ccagtgaatg tggcttattg ttttctggtc 36660 tatagaaaat tgtcgcaaactctgtcataa tgtctggttc tatataacaa agctagtcct 36720 gtattctgca tgtggctgatggaaacagtg ctctgttgat ctggttcatg aagaaatctg 36780 ttcaattctg cataacagatgccttcatca gtgtccttcc atgaaggagc tgatcttcac 36840 aaagaacaca tagttttgcatcccaccact tgcagtattt tttttttttt tttttttttt 36900 ttgagatgca gtctcgctctgtcaccctgg ctggagtgca gtggcatgat ctcagctcag 36960 tgcaacctct acctcctgggttcaattgat tctcctgcct cagcctcctg agtagctggg 37020 attacaggcg cacaccaccatgcctggcta atttttgttg ttttagtaga gacggagttt 37080 caccatattg gtcaggctggtctcaaactc ttgacctcat gatctgcctg ccttggcgtc 37140 ccaaagtgtt gggattacaggcgtgagtca ctgtgccctg ccagtattgt tttgtctaaa 37200 ttatttgtgc tgatgtttttcctactgtgg ttttcttcag attacccttg ctctgagcct 37260 gcaattgact catgaacttcttttccatgt tctaacctta caatgacttc cttgtgttca 37320 ctccaaatgt ttttccctggttgcatgtag agatgtatta gctaaggtac atgcttagct 37380 gctgtatcaa agagaccctaatgtacaacc caggctggta gagcagctct gctgtatgtg 37440 ttaattcagg gacccaggttccttccatgt tgtgactccc cccttcctta ggatgttgtc 37500 ttcttttaca tggctgaagttgggccattt catgtctctg ttccagctgc ctggtaggaa 37560 aaaagaacag aaattcagagtaagcaaatt ctttttctat agatggatgc ggaagttgga 37620 cacatcattt cctctcacattttctcggcc agaacgtagt catgtgactg cacgtctagc 37680 tgctaaggag actgggaatttactgtcggc tgtgtggcct ctgtcaagct aaaattctta 37740 ttactgtgga ataagggaaggatggatttg ggggcacaat taatagtctg tcacagaggc 37800 taaaacagct gcttttggctgggcacggtg gctcacacat gtaatttcag cactttggga 37860 ggccgaggca agtggatcacttgagatcag gaatttgaga ccagcctggc caacatggtg 37920 aaaccctgtc tctcctaaaaatatagaaat tagccgggca tggtggcggg tacctgtaat 37980 ccgagctact ccagaggttgaggcaggaga attgcttgaa cctggaaggc agaggttgca 38040 gtgagccaag atggtgccactgcactccag cctgggcgac agagcaagac tccatctcaa 38100 aaaaaaaaaa aaaggttaaataaacagctg cttttgtagg tgatacaagg tacagctaag 38160 ctttgaagcc aggcctgtagtttcaccttc catattctta ctcaaggcat tatacttctg 38220 gatctgaaac cactggatctgatgccctgc ttgggatgag ttctttatat tatcttgctt 38280 tcaacccaca cctgtgtaattttatgggca gcgtttgttt cctatatagg aacaatttga 38340 aagtgggctg tttctaggctttcatgaata gcaggctatg ctgtcattgg gaatctggag 38400 ggagttaatg aacacaacttcattgtttac tttagtgaaa tgtggcagct tatgatagtt 38460 ttgacagtga gacatgtgctgttttgatct ctcagctaag attatctgat ttttcaggca 38520 tgtctcaaaa ctcaccaggcctgctcacat gctgctgctt ctgaagccag ggtttggaaa 38580 ccagctgccc atcagaatgaggctgtgact tagaatattg gttcttgttt tattaccatt 38640 ccttgtttgg tctctccagagtcactggcc ttttccgctt caattttctt atcggtgaaa 38700 tgagatatta attcctcttattgacttcaa ttcaattgct gagtgtattg ttgcctttgg 38760 gaggttcttt gagttttctgtgcctttgaa atagttgttt ttttttattc tggtgttttg 38820 aggcatgttt caagtgagtgcatttacact tctaccattt taggagccac aattcagtta 38880 tgttgtccca gcttgcttggccccatcccc agagtttctg attcagtagg tctggggtgg 38940 ggcccaataa tttgcatttcttcttctttt ttcgagacag agtctgactg tgtcatccaa 39000 gctggagtgc agtggcacgatcgtagctca ttgtagcctc aaactcctgg gctcaagccg 39060 tcctcccacc tcaccctcctgagtagctgg gactataggc atatactacc atgccctgcc 39120 acctttttaa ttttttgtaaggatgggggt ctcactgtgt tgctcaggct ggtcttgaat 39180 tcctgggctg aagtgatcctcctgcttcag cctccccaaa tgccggcatt cctggcatga 39240 gccactgcac ttggccaagactttgcattt ctaactagtt tccaggtaat gctgctgctg 39300 gtgtagggac ctcattttgagaaccattgt tctatagctg tagctatagt tagtttctgg 39360 ttatagcttc ttccttttgtcccttcagta atagtgtaca catccgaaat ccctgtcctt 39420 gctctttcag gcccaggcatggtatctggt cctcttctgt tgctagccct ggggtgcttc 39480 atcatcccaa gtttatttttcttctcctaa cctgaacctt tgtaaatagc cccttcccta 39540 atgaacgtcc tcaattccctgttttgcgtg tcctgtctgt ttcttggcaa gactctggat 39600 gattcagtac tcaatgaggatttttcgcat agatggatga aacaggctgg gtttcatgtt 39660 ttctaagata aaggtgcttctctctttttc tcttggtcac tttgaccaag aagaaaataa 39720 cagagttttt attctcaagaagaataatat cggggccact ctgctcagag gccactctgc 39780 tttgaggacc ccttctctcctccctcatgc caaagatcag gaacattggg cagagcggat 39840 aacgatgccg ccagcgtcattacattttca cggcactttc agttgtgctg agcgtgcaaa 39900 catttcaagg agacatttctaagaggtggc tagcacagca tgcctctaat gccctatgtg 39960 aattggaata gagtactaaagaactgttca atattcaccc catccccgca tatgcaagca 40020 tgcacgtggg ttcattgtatatgtgtgtgt gcacgtgtgc acagacacat ttgtccttcg 40080 tttcaaatgc aacacaatggatggaaattg ccttcctggt actggggtat ggatgcaaac 40140 accaacagag aagcagccgctacttccaaa ctgaacacat gtgagatttg ccctttaatt 40200 agcatctgca gctgctgccatcagaagggt ctgtctctgt tggcctgaaa gtctttgctt 40260 taaaagagca agtccattatagctccaagc caggctcgtc tgtcagctgc tgtgctttct 40320 ctgccatcag cggggttgccacattgtttt gggctgtttc actctaggac tctttcctcc 40380 tcctgtgccc ccagcctttgattaccatgc cttggtgatc ctcatttggg tgacctgcag 40440 ctgctcattg tgtgtgcaggagacatctcc agtccttgta aggagggaag atcactggct 40500 tcagtgctga tggactggttattttccagc cctttgtcgt cagtgatctt gtcttgatat 40560 gcagaaaggc tccaggtagtcactgaaaaa aatataagca gcagaggtga tggctatatg 40620 aaagtcacgt ttcatcaagggcattgctgc tatggaaact ttcaattcac ttggagtagg 40680 gagccatatt ggttccacagcctcctcagc agtgggtccc aacacagtgc tgggctagct 40740 gcctctgaat caccgcagtagctcctttta ctatagattc ctgggtccca cccatggaat 40800 gtgatccatg aagtctggggttattccctg gaatccttta agctccctaa gtggttggga 40860 tgggaaagag atatgctttatgttactata cttcttctta ttattatttt aaaattcttg 40920 ccgggcgcag tggctcacacctgtaatccc agcacattgg gagaccgagg cgggtggatc 40980 acttgaggtc aggagttcgagactggcctg gccaacatga tgaaatcccg tctctactaa 41040 aaatacaaaa attagctgggcatggtggcg catgcctgta gtcccagcca ctccggaggc 41100 tgaggcagga gaatcgcttgaacccgggag gcagaggttg cagtgagccg agatcgtggc 41160 actgcactcc agcctgggtaacagagtgag acttcatctc aaaaaaaacc caaaaaaaca 41220 aaactctttt tcattataccggaacgtcag ctttatggag tcggggattt tttctgtttt 41280 attcactgct gtttccctaacatctagaat agtggctggc acgataggca ctcaagtatt 41340 gatttagatg agtctattttattttctttt aaatttttaa tttttattag aggtggggtc 41400 tggctttgtt gcccaagctggtctcaaaac tcctggcctc aagcgattgt actgcctcag 41460 cctcccaaag ggctaggataggcatgagcc aacatgcctg gcttgtctta tttttaacaa 41520 gcacttctgg tgattctgatggacaatcag gcttgggaag ttctaaccta gaggacctac 41580 agttgtcttg gggtagaagccaaggctatc ctggttttta gaatcagtgc cttactgggc 41640 atctctgaag agtaaaagtcagggacagag ttacattttt ggacaaaacc agatgctgtg 41700 aatggactct tggtcacaacctgggtggcg acttggtcct taacttcttc atcattttct 41760 gctgaccctg ttctttggttcacagcaagt cacctgataa gaagactcaa agactgctag 41820 tttgttactt tagatgatgcttttggaacc tcttggtacc attttaacaa tccaaacgta 41880 ttttatgaaa gcactcaagtcctgggtctt tattgtatct ttaagctcta acagcatgat 41940 gattgaataa gctgtggttggccacacaca agccatcttc cccatggcct ccattcatac 42000 tagaatgagc agctataccccagtagtata gttttgggat atgggtaaca tcttgggata 42060 gccacattta cttagtaaatgtctggctta cattctccta atggtgcact gttggaattt 42120 ttggtgtggt aacctggaatagtgttggtg ggtcaagttt gattagcatc tttgataagg 42180 acccggtcta tttagaggtttgtcattgag tgtgtctgtt ttggcctcat gttgtgaagc 42240 atgctgtgta gcagctgttgtaatttttgt tgcttgtttt ctcaatcaac cctggttttg 42300 aagaaatggg aagttgttccactcttagac tgatctgact tgggagggga ttttcagttc 42360 aggaagttgg atcttctgaatggaagcaaa gaatacatgt ctttttgcca ctttacaagc 42420 tggctcttgt tttctgaactattttactgg tcattgcaaa tagaatgtca ggagtagctg 42480 ccaaatacta agttgtgttcagtttgtcag ttcttaagag ttgccggtgg ctgctctgct 42540 atgcgtatga ctttctcagccttaaactta caagccatac tgtttttttc acatctttaa 42600 tacagccata ggaaatttataactgtggcg tgtcgtcata aatatgcatt gttcttattt 42660 taagacattt cagtactaaaagtataagta cttctgttat tatctgtgaa tttctttcct 42720 tcttcttttt ttggatatttaagacctttt cgatgtcaat atatatttaa aacagacata 42780 taaattagca ttcacccacatacccagggc ctatggagaa ccaggttggg atgagtgggt 42840 gagctacagg cagccaggtggctcctgtgg gctcctcgag gactggggtg agtaactaat 42900 gtctgctagg aacttgggggaaagaaggtg tgtatgttag gtgctgcccc cttctaagtg 42960 ttcctcttgt tcataatttttttttttttt ttttttttta gatggagtct cgctctgttg 43020 ccaggctgga gtgcagtggtgtgatctcag ctcactgcaa cctctgcctc ccgggttcaa 43080 gtgattctcc tgcctcagcctcccgagtag ctgggactac aggcatgcac caccatgccc 43140 agctaatttt tgtatttttagtaaagacgg ggtttcacca tgttggccag ggtggtctcg 43200 atctcttgac cttgtgatccgcctgcctcg gcctcccaaa gtgctgggat tacaggtgtg 43260 agccactgtg cccagcccataaatcaaaat tttttcagca attgttatac aagtggaacc 43320 ttactcttca aatgcaattgtccagtgtct ggcttaatgt ctgctgttgt cagaaaccat 43380 gtgaatggag tagattcccaggttataagg agcccccagg gaggatgcgc gagtcactgg 43440 cttctccagg ggtctctggtttggggttgc cttggtgctg ggcacacttc ctggagattt 43500 tactggacca gcctgaggcctttggggctc tgtgcagatg ctctacttct gacttgtcta 43560 gagctttctt ctaattctggactaaaagca agcaggagtt tggaggatga tggtgagaat 43620 tcacatcccc gagttggcttttggaatgca gtagtttgtg agatttagtg ttttttttaa 43680 gaagtatatt cagatcttgcctttttccca gaaagcatat gagacaactt ccaagacatt 43740 tatagcatgg ctaataaaatgggaaatcag ggcgaaggac aggagaactc aataagggtt 43800 aacatggcta cagcgattgtctaaatgggt tctttttgct ggccagagca gaaaggatca 43860 tgcagtaaag tgggggggaagaaagggaat tgaatggtag gtgaagactt catgttggtg 43920 ccaggcactg tgccaggccctcctaggacc ttgtcttact caatcctcac acagtgctgc 43980 aagaggatta gtcttatccctgttttagag aggatgaaac tgaaaggcag cgaggtgaag 44040 tcaccagcag gaggctgaagccgcccaggc taactggcct tatagctacc tagggactca 44100 ggaatatcac acctgtttatcatcaaaagg agaaaggatt tcagttcctt ggggtagaag 44160 agtttctttt tgctaatcaaacattttact tgaggcttca tattcttctt caagattttt 44220 ttcctgtgta tgtaccaacacatgtaataa ttccttgttt atttcaaaaa aggggttgta 44280 ctttattctt tacaagatttcactttatat tgtcatggac aattttccat ggcagtatga 44340 ataaatggaa tctgtttgtttttaatatct ttgtcttatc ccattgttta catatgtcat 44400 attttagcca gtctctaactgatggatagc tgaatgattt ccatgttttt ttcccctgtt 44460 acaaacaata ctgcaaggaatctatttatc tttctattta tctgcaaact attgtaagta 44520 cctgtaaatt gttagaagtggaattactag gtcaaagggg atattttcac atttaaattt 44580 tgaatagagg ctgtcagttgccttccacac tgactataaa aggaaaagat tgtatcacat 44640 ttattgcaag ccttctgtattctgctgggt gctgagggga atacagaaag gatataagag 44700 tggttgccct ctaggaatatccgtctacac tgtacctaat cctagggaat gtctggggtg 44760 tcaacttgtg ggtgggaaagtgggtggatt taattcaact gttcaagctt gccttgcaaa 44820 cactgtgcat ggtgtctgggactagtcttt cattatattg attcccctgg gtaacagatg 44880 taatttcctt agggcagggacttcatccta catgacttac agcgtgcctt acacatcttc 44940 tttgctttgt ggagaccttgttattataac acgtcaggtg atattcgagg atctaattga 45000 ggcattccct atttttgggtgtgtgaagaa ttaataactt tggcattcta tacaggtcat 45060 ggaatatcag cctggaggggacttgctgtc acttttgaat agatatgagg accagttaga 45120 tgaaaacctg atacagttttacctagctga gctgattttg gctgttcaca gcgttcatct 45180 gatgggatac gtgcatcggtaagtgagact ctggtagcat ttttatgctg aggattttcc 45240 tgtgtcgcat aagagttcctgcatggaaat gagtggatga gtgatttcaa gatcaagata 45300 acgccccatc cagtttttagccagtctacc aataactggc tgaaagcaaa ctttccaaga 45360 tggaggacat ttcagcttgcttatccagca gtgcaataga tctagaattg taatgtgctc 45420 aagtttgcta gtaatatctattaatgtagc taaataagac tgggaactct tgcatgggtt 45480 ctttgggtta tatgatagaagaactgaatt tggtttgcag aaggaaatgt cataccacat 45540 agtagtgtaa gaccatggagctgtacttct ctaactctgc ccgttagaat ttacaatttt 45600 tttttttttt tttttttttgagacagagtc tagctctgtt gccaggctgg agtgcagtgg 45660 taccatattg gctcatggcaacctccgcct cctgggttca ggtgattctc ctgcctcagc 45720 ctcccaagta gctggaattacaggcacgca ccgccatgcc cagctaattt ttgtattttt 45780 agtagagatg gggtttcaccaggttggcca ggatggtctt gatctcctga cctcatgatc 45840 cacccacctt gtcctcccaaattgctggga ttacaggcat gagccaccat gcctggccta 45900 caaaatcctc agttggtaagtggttcttca tgtcttcatt catctgatgt tttgtgtaca 45960 tctgagaatg ttgtgggaatacaatgattg ttagtccagg aatcacaaaa tttgagatag 46020 agtctcagct tttccattgcctagctacat gaccttggga aaatttcata gctccttttg 46080 gccttagttt tcctcatgtgaaatgtgtgt ctctaggaga aataatccat tgaataatat 46140 gtgtttcatt tctcttccttttctttctct cctatccttc cttgctccct ctcgcccttt 46200 ttctctttcc ccctctctccctctctctct ccttccttcc ttcctttcgg ttaaattcat 46260 tttgcaaaat gtatgctaataatttatatc caccaataga ggaggtctat ataacagaat 46320 acataaacaa agatttttggctcaattgag attctaggtt agcacttgct tgctgattgg 46380 gatggaggag gcaattcatggtcctgattt tcttacagag acatcaagcc tgagaacatt 46440 ctcgttgacc gcacaggacacatcaagctg gtggattttg gatctgccgc gaaaatgaat 46500 tcaaacaaga tggtaaaaaatggaataaga tagcttaata gagtttatac taaaaagtat 46560 tcttggtcct cctaagtttgggaagtgttg ggataaaatg gtgaacaatg ttttggagcc 46620 tttggcagtg tatgggggtggggacaggga cacagaacca tttcccagac cgtggcacct 46680 ttttatttat agtgcctgttaataccctcc aagacatttt taggagcatt gttatagttt 46740 ggttagaaat aaaggaaaatgcttattttg tttctctctt cattttcctt gcctgttata 46800 gactgtcttt tgttatattatcttttttac tttaaaatat tttgatgaaa tggaaactcc 46860 tgcatgtcaa atcctctatttcctatgcag caaaattgaa attaatcact ggagcatttg 46920 aaccaaatat ccttaagtgttaagaaccaa gtgctcaaaa tatcattttt aagtcttgga 46980 tctttggtag aaattaaactgtattccaca tgctaagtag gacggcagga gggtagctac 47040 tgagatcaag agtgagactactttaggaaa aagatgacaa agtaaaaaaa gattagagtt 47100 taaaaatctt ctaataaagttggtatgtac taaaatatga atttggaagt caactccgca 47160 aaaaaggata ggtctaagagaaaatcgact taggttttaa gactgatttt acaactgagc 47220 catttggtga cctagacaaatccttgggaa cttgatcttt tatactttct ctagaaaaaa 47280 ctgatgctag tgaaaatgcataatttaaga ggttagagaa gctgctcttc aaaatgcccc 47340 ccaagtctga gagttaaatcctttacataa aggacaatat gtaaaatttt ctttttcttt 47400 tttctttttt tttgagacggagtctcgctc tgtcccccag gctggagtgc agtggcgcga 47460 tctcggctca ctgcaagctccgcccccctg ggttcacgcc attctcctgc ctcagcctcc 47520 cgagtagctg ggactgcaagcgcccgccac catgcccagc taattttttg tatttttagt 47580 agagacgggg tttcaccgtgttagccagga tggtctcgat ctcctgacct cgtgatccac 47640 tcgcttcggc ctcccaaagtgctgggatta caggcataag ccactgcgcc cggctctttt 47700 ttttcttaaa ctgcttccagaaaagtggat attattaggt tgatgttaag aaaaggcttg 47760 gagttgcatt aactttttgctttctagcat ctggcctgtc tgttctgcag acctgagacc 47820 tacttgagat aattttcttggtgttcaggc ccttggaaaa ataagttccc tatgttgtcc 47880 agtgtcaaag tttctcaacctcagcactat tctttttttc aggttatttt cttgtaatct 47940 gttcacttga tcattacattaagaattaga ttatattgct ataactacaa agcattttat 48000 gttttaaaaa ttatgtacaatttagaaaca ggcatgaaaa cttaggtatt aaatttagtg 48060 gaataaagca cagaaaaaaagttaaaataa tgcagtttta tcacttagga ttaaacattt 48120 atatgggccg ggtgtagtgcctcacacctg taatcccagc acgtttggag gtcgaggcgg 48180 gaggattgct ggagtttgagaccagcctgg gcaacaaaat gagacctagt ctctacaaaa 48240 aatcaaaaaa ttagccagacatggtagtac atgcttgtag ctccagccac atgggaggcc 48300 aagacagtag gatcgctggagcgaaggagg ttgaggctgc aatgaccgtg tttgcaccat 48360 tgcattccag cctgggcgacagaacaagac cctgtcttaa aacaaattta tatgctgcat 48420 tcgtgaaatt aaaaaaaaatcatggattta gaaataaatt gaagcaaggt acattgacag 48480 tgtaacctca gcactactgacattttgatc tgaataattc tttgttgtgg gggatgcgct 48540 gtataagatg tttagctgcatccctgactc ctacctccta gatgccatta gcaccctccc 48600 ctccagatgt gataaccaaaaatgtctcta gacattgcca gatgtgcctg gggtaggagg 48660 gttgggggaa gtggggtttgagaaccctta gttgatcatg cctgcagtag gttgagaagc 48720 atcagaaagc taattaattagacaggaata tgtgtttgca gta 48763 4 251 PRT Rattus norvegicus 4 Met LeuLys Phe Lys Tyr Gly Val Arg Asn Pro Ser Glu Ala Ser Ala 1 5 10 15 ProGlu Pro Ile Ala Ser Arg Ala Ser Arg Leu Asn Leu Phe Phe Gln 20 25 30 GlyLys Pro Pro Leu Met Thr Gln Gln Gln Met Ser Ala Leu Ser Arg 35 40 45 GluGly Val Leu Asp Ala Leu Phe Val Leu Leu Glu Glu Cys Ser Gln 50 55 60 ProAla Leu Met Lys Ile Lys His Val Ser Ser Phe Val Arg Lys Tyr 65 70 75 80Ser Asp Thr Ile Ala Glu Leu Arg Glu Leu Gln Pro Ser Val Arg Asp 85 90 95Phe Glu Val Arg Ser Leu Val Gly Cys Gly His Phe Ala Glu Val Gln 100 105110 Val Val Arg Glu Lys Ala Thr Gly Asp Val Tyr Ala Met Lys Ile Met 115120 125 Lys Lys Ala Ala Leu Arg Ala Gln Glu Gln Val Ser Phe Phe Glu Glu130 135 140 Glu Arg Asn Ile Leu Ser Gln Ser Thr Ser Pro Trp Ile Pro GlnLeu 145 150 155 160 Gln Tyr Ala Phe Gln Asp Lys Asn Asn Leu Tyr Leu ValMet Glu Tyr 165 170 175 Gln Pro Gly Gly Asp Leu Leu Ser Leu Leu Asn ArgTyr Glu Asp Gln 180 185 190 Leu Asp Glu Asn Met Ile Gln Phe Tyr Leu AlaGlu Leu Ile Leu Ala 195 200 205 Val His Ser Val His Gln Met Gly Tyr ValHis Arg Asp Ile Lys Pro 210 215 220 Glu Asn Ile Leu Ile Asp Arg Thr GlyHis Ile Lys Leu Val Asp Phe 225 230 235 240 Gly Ser Ala Ala Lys Met AsnSer Asn Lys Val 245 250 5 251 PRT Mus musculus 5 Met Leu Lys Phe Lys TyrGly Val Arg Asn Pro Pro Glu Ala Ser Ala 1 5 10 15 Ser Glu Pro Ile AlaSer Arg Ala Ser Arg Leu Asn Leu Phe Phe Gln 20 25 30 Gly Lys Pro Pro LeuMet Thr Gln Gln Gln Met Ser Ala Leu Ser Arg 35 40 45 Glu Gly Met Leu AspAla Leu Phe Ala Leu Phe Glu Glu Cys Ser Gln 50 55 60 Pro Ala Leu Met LysMet Lys His Val Ser Ser Phe Val Gln Lys Tyr 65 70 75 80 Ser Asp Thr IleAla Glu Leu Arg Glu Leu Gln Pro Ser Ala Arg Asp 85 90 95 Phe Glu Val ArgSer Leu Val Gly Cys Gly His Phe Ala Glu Val Gln 100 105 110 Val Val ArgGlu Lys Ala Thr Gly Asp Val Tyr Ala Met Lys Ile Met 115 120 125 Lys LysLys Ala Leu Leu Ala Gln Glu Gln Val Ser Phe Phe Glu Glu 130 135 140 GluArg Asn Ile Leu Ser Arg Ser Thr Ser Pro Trp Ile Pro Gln Leu 145 150 155160 Gln Tyr Ala Phe Gln Asp Lys Asn Asn Leu Tyr Leu Val Met Glu Tyr 165170 175 Gln Pro Gly Gly Asp Phe Leu Ser Leu Leu Asn Arg Tyr Glu Asp Gln180 185 190 Leu Asp Glu Ser Met Ile Gln Phe Tyr Leu Ala Glu Leu Ile LeuAla 195 200 205 Val His Ser Val His Gln Met Gly Tyr Val His Arg Asp IleLys Pro 210 215 220 Glu Asn Ile Leu Ile Asp Arg Thr Gly Glu Ile Lys LeuVal Asp Phe 225 230 235 240 Gly Ser Ala Ala Lys Met Asn Ser Asn Lys Val245 250 6 251 PRT Mus musculus 6 Met Leu Lys Phe Lys Tyr Gly Val Arg AsnPro Pro Glu Ala Ser Ala 1 5 10 15 Ser Glu Pro Ile Ala Ser Arg Ala SerArg Leu Asn Leu Phe Phe Gln 20 25 30 Gly Lys Pro Pro Leu Met Thr Gln GlnGln Met Ser Ala Leu Ser Arg 35 40 45 Glu Gly Met Leu Asp Ala Leu Phe AlaLeu Phe Glu Glu Cys Ser Gln 50 55 60 Pro Ala Leu Met Lys Met Lys His ValSer Ser Phe Val Gln Lys Tyr 65 70 75 80 Ser Asp Thr Ile Ala Glu Leu ArgGlu Leu Gln Pro Ser Ala Arg Asp 85 90 95 Phe Glu Val Arg Ser Leu Val GlyCys Gly His Phe Ala Glu Val Gln 100 105 110 Val Val Arg Glu Lys Ala ThrGly Asp Val Tyr Ala Met Lys Ile Met 115 120 125 Lys Lys Lys Ala Leu LeuAla Gln Glu Gln Val Ser Phe Phe Glu Glu 130 135 140 Glu Arg Asn Ile LeuSer Arg Ser Thr Ser Pro Trp Ile Pro Gln Leu 145 150 155 160 Gln Tyr AlaPhe Gln Asp Lys Asn Asn Leu Tyr Leu Val Met Glu Tyr 165 170 175 Gln ProGly Gly Asp Phe Leu Ser Leu Leu Asn Arg Tyr Glu Asp Gln 180 185 190 LeuAsp Glu Ser Met Ile Gln Phe Tyr Leu Ala Glu Leu Ile Leu Ala 195 200 205Val His Ser Val His Gln Met Gly Tyr Val His Arg Asp Ile Lys Pro 210 215220 Glu Asn Ile Leu Ile Asp Arg Thr Gly Glu Ile Lys Leu Val Asp Phe 225230 235 240 Gly Ser Ala Ala Lys Met Asn Ser Asn Lys Val 245 250

That which is claimed is:
 1. An isolated nucleic acid moleculeconsisting of a nucleotide sequence selected from the group consistingof: (a) a nucleotide sequence that encodes a polypeptide comprising theamino acid sequence of SEQ ID NO:2; (b) a nucleotide sequence consistingof SEQ ID NO:1; (c) a nucleotide sequence consisting of SEQ ID NO:3; and(d) a nucleotide sequence that is completely complementary to anucleotide sequence of (a)-(c).
 2. A nucleic acid vector comprising thenucleic acid molecule of claim
 1. 3. A host cell containing the vectorof claim
 2. 4. A process for producing a polypeptide comprisingculturing the host cell of claim 3 under conditions sufficient for theproduction of said polypeptide, and recovering said polypeptide.
 5. Anisolated polynucleotide consisting of the nucleotide sequence set forthin SEQ ID NO:1.
 6. An isolated polynucleotide consisting of thenucleotide sequence set forth in SEQ ID NO:3.
 7. A vector according toclaim 2, wherein said vector is selected from the group consisting of aplasmid, a virus, and a bacteriophage.
 8. A vector according to claim 2,wherein said isolated nucleic acid molecule is inserted into said vectorin proper orientation and correct reading frame such that a polypeptidecomprising SEQ ID NO:2 may be expressed by a cell transformed with saidvector.
 9. A vector according to claim 8, wherein said isolated nucleicacid molecule is operatively linked to a promoter sequence.